Encochleation methods, cochleates and methods of use

ABSTRACT

Disclosed are novel methods for making cochleates and cochleate compositions that include introducing a cargo moiety to a liposome in the presence of a solvent. Also disclosed are cochleates and cochleate compositions that include an aggregation inhibitor, and optionally, a cargo moiety. Additionally, anhydrous cochleates that include a protonized cargo moiety, a divalent metal cation and a negatively charge lipid are disclosed. Methods of using the cochleate compositions of the invention, including methods of administration, are also disclosed.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/822,230 filedApr. 9, 2004, which claims the benefit of and priority to U.S.Provisional Application No. 60/461,483 filed Apr. 9, 2003; U.S.Provisional Application Ser. No. 60/463,076, filed Apr. 15, 2003; U.S.Provisional Application Ser. No. 60/502,557, filed Sep. 11, 2003; U.S.Provisional Application Ser. No. 60/537,252, filed Jan. 15, 2004; U.S.Provisional Application No. 60/499,247 filed Aug. 28, 2003; U.S.Provisional Application No. 60/532,755, filed Dec. 24, 2003; and U.S.Provisional Application No. 60/556,192, filed Mar. 24, 2004. The entirecontents of each of the aforementioned applications are hereby expresslyincorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

Portions of the subject matter disclosed herein were supported byFederal Grant No. NIAID SBIR PI R43 AI46040-01, awarded by the NationalInstitutes of Health. The U.S. Government may have certain rights in theinvention.

TECHNICAL FIELD

The invention generally relates to cochleate delivery vehicles. Morespecifically, the invention relates to novel methods of making and usingcochleates employing a solvent to encochleate a cargo moiety, tocochleates including one or more aggregation inhibitors, and tocochleates including a protonized cargo moiety, divalent cation andnegatively charged lipid.

BACKGROUND

The advantages of cochleates are numerous. For example, cochleates aremore stable than aqueous structures such as liposomes, they can bestored lyophilized which provides the potential to be stored for longperiods of time at room temperatures, they maintain their structure evenafter lyophilization (whereas liposome structures are destroyed bylyophilization), and they are non-toxic.

Cochleate structures have been prepared first by D. Papahadjopoulos asan intermediate in the preparation of large unilamellar vesicles. U.S.Pat. No. 4,078,052. Methods of making and using cochleates to deliver avariety of molecules have been disclosed, e.g., in U.S. Pat. Nos.5,994,318 and 6,153,217.

In these methods, prior to precipitation of the cochleates, the materialto be encochleated is introduced into liposomes by solubilization of thelipid and material in solvent, removal of the solvent to form a drylipid film, then by hydration of the lipid and components to beencochleated. Alternatively, material and lipid may be solublized indetergent which may be removed by dialysis or other methods. These stepsare time consuming, represent added expense in manufacturing and productcosts, and can in some instances affect the activity and/or stability ofthe encochleated material.

Additionally, cochleates are highly susceptible to aggregation, and thusparticle size and particle size distribution can be highly variable andunstable after preparation and removal from the two-phase polymersystem. The ability of drugs to be administered via the oral routedepends on several factors. The drug typically must be sufficientlysoluble in the gastrointestinal fluids in order for the drug to betransported across biological membranes for an active transportmechanism or have a sufficiently small particle size, such that it canbe absorbed through the Peyer's Patches in the small intestine andthrough the lymphatic system. Particle size is an important parameterwhen oral delivery is to be achieved (see Couvreur P. et al, Adv. DrugDelivery Reviews 10:141-162, 1993). Thus, it would be advantageous to beable to control and stabilize the particle size and particle sizedistribution of encochleated materials.

There also exists a need for delivery vehicles that can safely andeffectively deliver cargo moieties that are poorly absorbed by the body(e.g., weakly basic drugs). For example, aminoglycopeptides (e.g.,vancomycin), are poorly absorbed through the GI tract and are difficultto deliver to cells harboring bacteria. Accordingly, in order toadminister an effective amount of drug against a bacterial infection,large amounts of drug are ingested to not only account for poorabsorption through the GI tract, but also poor delivery to the site ofinfection. Consequently, a toxic level of drug can accumulate in the GItract (e.g., in the kidneys) or the blood stream and can lead to seriousillness, such as erythematous or urticarial reactions, flushing,tachycardia, and hypotension. Aminoglycosides (e.g., streptomycin andtobramycin) are similarly problematic because of the risk ofnephrotoxicity and ototoxicity due to poor absorption, which can lead toacute, renal, vestibular and auditory toxicity. While these drugs can bedelivered intravenously to bypass the issue of poor GI tract absorption,uptake by the cells is still problematic. That is, even at highconcentrations, aminoglycopeptides and aminoglycosides can not penetratethe cell membrane in order to contact the bacteria. Additionally,echinocandins (e.g., caspofungin), a new, less toxic class of antifungaldrugs, still have unwanted side effects and poor oral bioavailability.As such, they are generally administered intravenously.

The present invention addresses each of these drawbacks.

SUMMARY OF THE INVENTION

The present invention provides novel methods of forming cochleates,which methods can be efficiently and easily scaled up. Additionally, thepresent invention provides an anhydrous cochleate including a protonizedcargo moiety, e.g., an aminoglycoside, and methods for making andadministering the same. The present invention also provides a cochleatecomposition which includes an aggregation inhibitor, and methods formaking an administering the same.

In one aspect, the present invention provides a method for forming acargo moiety-cochleate, which includes introducing a cargo moiety to aliposome in the presence of a solvent such that the cargo moietyassociates with the liposome and precipitating the liposome to form acargo moiety-cochleate. The cargo moiety can be any cargo moietydescribed herein, including protonized cargo moieties. In certainembodiments, the cargo moiety is hydrophobic, hydrophilic, hydrosolubleor amphipathic. In other embodiments, the cargo moiety is an antifungalagent.

In preferred embodiments, the solvent is a water miscible solvent, morepreferably the solvent is dimethylsulfoxide (DMSO), a methylpyrrolidone,N-methylpyrrolidone (NMP), acetonitrile, alcohol, ethanol,dimethylformamide (DMF), ethanol (EtOH), tetrahydrofuran (THF), orcombinations thereof. In certain embodiments, the method can furtherinvolve introducing a solution of the solvent and the cargo moiety to anaqueous liposomal suspension. In preferred embodiments, the solution isintroduced dropwise, by continuous flow addition, or in a bolus.Additionally or alternatively, the method can further involveintroducing the cargo moiety to a liposomal suspension comprising thesolvent. In preferred embodiments, the cargo moiety is introduced as apowder or a liquid. In other embodiments, an antioxidant can beintroduced to the liposomal suspension.

In yet other embodiments, the liposomal suspension comprises a pluralityof unilamellar and multilamellar liposomes. In preferred embodiments,the method additionally includes the step of filtering or mechanicallyextruding through a small aperture the liposomal suspension such that amajority of the liposomes are unilamellar.

In still other embodiments, the method further involves precipitatingthe liposome with a multivalent cation to form a cargo moiety-cochleate.In yet other embodiments, the solvent can be removed from the liposomeby dialysis and/or washing.

In some embodiments, the ratio of the lipid to the cargo moiety isbetween about 0.5:1 and about 20:1. In other embodiments, the ratio ofthe lipid to the cargo moiety is between about 20:1 and about 20,000:1.

In other embodiments, the method also includes introducing anaggregation inhibitor to the liposomes or the cochleates. Theaggregation inhibitor can be any aggregation inhibitor described herein.

In another aspect, the instant invention provides composition whichincludes one or more cochleates made by any one of the methods describedherein.

In yet another aspect, the instant invention provides a compositionincluding an anhydrous cochleate. In one embodiment, the anhydrouscochleate includes a negatively charged lipid, a protonized cargomoiety, and a divalent metal cation. In some preferred embodiments, theprotonized cargo moiety is water soluble. In other preferredembodiments, the protonized cargo moiety is a protonized weakly basiccargo moiety. In still other preferred embodiments, the protonized cargomoiety is a multivalent cation.

In particularly preferred embodiments, the protonized cargo moiety is aprotonized peptide, a protonized protein, a protonized nucleotide,including a protonized DNA, a protonized RNA, a protonized morpholino, aprotonized siRNA molecule, a protonized ribozyme, a protonized antisensemolecule, or a protonized plasmid, an aminoglycoconjugate, such as aprotonized aminoglycoside or a protonized aminoglycopeptide, includingprotonized vancomycin, teicoplanin, bleomycin, peptidolglycan,ristocetin, sialoglycoproteins, orienticin, avaporcin, helevecardin,galacardin, actinoidin, gentamycin, netilmicin, tobramycin, amikacin,kanamycin A, kanamycin B, neomycin, paromomycin, neamine, streptomycin,dihydrostreptomycin, apramycin, ribostamycin, spectinomycin, or aprotonized echinocandin, including protonized caspofungin, echinocandinB, aculeacin A, micafungin, anidulafungin, cilofungin, pneumocandin andany combinations thereof.

In some embodiments, the ratio of protonized cargo moiety to lipid isabout 2:1 by weight. In other embodiments, the ratio of protonized cargomoiety to lipid is between about 4:1 and about 10:1 by weight. In yetother embodiments, the composition can additionally include a secondprotonized cargo moiety or a cargo moiety. The cargo moiety nay be anyof the cargo moieties discussed herein. In a preferred embodiment, thecargo moiety is a nutrient. In a particularly preferred embodiment, thenutrient is Vitamin E. In other preferred embodiments, the divalentmetal cation is barium or calcium.

In some embodiments, the composition may include an aggregationinhibitor. Any of the aggregation inhibitors discussed herein may beused.

In some embodiments, the lipid may include a phospholipid. In preferredembodiments, the phospholipid is a dioleoylphosphatidylserine (DOPS)and/or a phosphatidylserine (PS).

In still other embodiments, the present invention provides a method forforming an anhydrous cochleate which includes the step of contacting anegatively charged lipid, a protonized cargo moiety and a divalent metalcation, such that a cochleate is formed.

In some preferred embodiments, the method includes the step ofacidifying a cargo moiety to form a protonized cargo moiety. In otherpreferred embodiments, the method includes the step of adjusting the pHof a solution of the cochleate to maintain a protonized cargo moiety.

In yet other embodiments, the cochleate further comprises a secondprotonized cargo moiety. In still other embodiments, the divalent metalcation is barium or calcium.

In still other embodiments, an aggregation inhibitor can be introducedto the cochleate. In preferred embodiments, the aggregation inhibitor isintroduced to the cochleate before and after the cochleate is formed. Inparticularly preferred embodiments, the aggregation inhibitor comprisescasein and methylcellulose, and the casein is introduced before thecochleate is formed and the methylcellulose is introduced after thecochleate is formed.

In another aspect, the present invention is directed to a cochleatewhich includes an aggregation inhibitor. In certain embodiments, thepresent invention is directed to a cochleate composition including aplurality of cochleates and an aggregation inhibitor. In some preferredembodiments, the aggregation inhibitor coats the cochleate. Thecochleate composition can further include a cargo moiety, and the cargomoiety can be any of the cargo moieties discussed herein, includingprotonized cargo moieties. In preferred embodiments, the cochleateincludes an antifungal drug. Preferred aggregation inhibitors includeproteins, peptides, polysaccharides, milk or milk products, polymers,gums, waxes and/or resins. Particularly preferred aggregation-inhibitorsinclude casein, κ-casein, milk, albumin, serum albumin, bovine serumalbumin, rabbit serum albumin, methylcellulose, ethylcellulose,propylcellulose, hydroxycellulose, hydroxymethyl cellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose,polyvinyl pyrrolidone, carboxymethyl cellulose, carboxyethyl cellulose,pullulan, polyvinyl alcohol, sodium alginate, polyethylene glycol,polyethylene oxide, xanthan gum, tragacanth gum, guar gum, acacia gum,arabic gum, polyacrylic acid, methylmethacrylate copolymer, carboxyvinylpolymer, amylose, high amylose starch, hydroxypropylated high amylosestarch, dextrin, pectin, chitin, chitosan, levan, elsinan, collagen,gelatin, zein, gluten, carrageenan, carnauba wax, shellac, latexpolymers, milk protein isolate, soy protein isolate, and/or whey proteinisolate. In particularly preferred embodiments, the aggregationinhibitor is casein, methylcellulose, albumin, serum albumin, bovineserum albumin and/or rabbit serum albumin.

In preferred embodiments, the plurality of cochleates has a meandiameter of less than about 600 nm. In particularly preferredembodiments, the plurality of cochleates has a mean diameter of lessthan about 500 nm. In other preferred embodiments, the size distributionof the plurality of cochleates is less than about 700 nm. Inparticularly preferred embodiments, the size distribution of theplurality of cochleates is less than about 550 nm.

In other embodiments, the cochleate further includes an antifungal drug.In preferred embodiments, the antifungal drug is Amphotericin B,miconazole nitrate, ketoconazole, itraconazole, fluconazole,griseofulvin, clotrimazole, econazole, terconazole, butoconazole,oxiconazole, sulconazole, saperconazole, voriconazole, ciclopiroxolamine, haloprogin, tolnaftate, naftifine, terbinafine hydrochloride,morpholines, flucytosine, natamycin, butenafine, undecylenic acid,Whitefield's ointment, propionic acid, caprylic acid, clioquinol,nystatin, selenium sulfide, caspofungin, echinocandin B, aculeacin A,micafungin, anidulafungin, cilofungin, and/or pneumocandin. Inparticularly preferred embodiments, the antifungal drug is AmphotericinB and the aggregation inhibitor comprises methylcellulose. In otherparticularly preferred embodiments, the composition is a nasal spray.

In yet another aspect, the present invention provides a cochleatecomposition which includes a first plurality of cochleates with a firstmean particle size and a second plurality of cochleates with a secondmean particle size, wherein the second mean particle size is differentfrom the first mean particle size. In preferred embodiments, thecomposition includes at least one cargo moiety. In some particularlypreferred embodiments, the first plurality of cochleates and the secondplurality of cochleates include the same cargo moiety. In otherparticularly preferred embodiments, the first plurality of cochleatescontains a different cargo moiety than the second plurality ofcochleates.

In another embodiment, the cochleate composition can include a thirdplurality of cochleates with a third mean particle size, wherein thethird mean particle size is different from both the first and the secondmean particle sizes. In a preferred embodiment, the third plurality ofcochleates includes a cargo moiety.

In still another aspect, the present invention provides a method ofmaking a cochleate composition including introducing an aggregationinhibitor to a cochleate composition. In some embodiments, the methodincludes introducing the aggregation inhibitor to a composition ofcochleates. In other embodiments, the method includes introducing theaggregation inhibitor to a composition of aggregated cochleates. Instill other embodiments, the method includes introducing the aggregationinhibitor to a composition of liposomes and inducing formation of thecochleate composition. In yet other embodiments, the method includesintroducing the aggregation inhibitor to a solution of lipids, forming aliposomes, and inducing formation of the cochleate composition. Inpreferred embodiments, the aggregation inhibitor is added in anaggregation inhibitor to lipid ratio of between about 4:1 and about0.1:1 by weight. In particularly preferred embodiments, the aggregationinhibitor is added in an aggregation inhibitor to lipid ratio of about1:1 by weight. In other particularly preferred embodiments, theaggregation inhibitor is added in an amount suitable for modulating theresulting cochleate to the desired size range.

In yet other embodiments, the present invention includes pharmaceuticalcompositions including any of the cochleates or cochleate compositionsdiscussed herein.

In still other aspects, the present invention provides methods fortreating a subject that can benefit from the administration of a cargomoiety, including protonized cargo moieties, by administering cochleatesor cochleate compositions such that the cargo moiety is administered tothe subject and such that the subject is treated. Cochleates orcochleate compositions of the invention can be made using any of themethods described herein, including introducing a cargo moiety to aliposome in the presence of a solvent such that the cargo moietyassociates with the liposome and precipitating the liposome to form acargo moiety-cochleate. Any of the cargo moieties and protonized cargomoieties described herein can be administered in the cochleates of thepresent invention. In another preferred embodiment, the cochleatecompositions include an aggregation inhibitor. In particularly preferredembodiments, the aggregation inhibitor is casein, methylcellulose,albumin, serum albumin, bovine serum albumin and/or rabbit serumalbumin.

In a preferred embodiment, the cochleates are used for treating abacterial infection in a host. In other preferred embodiments, thecochleates are used for treating a fungal infection in a host. Inparticularly preferred embodiments, the host of the bacterial infectionor the fungal infection is a cell, a tissue or an organ. In otherpreferred embodiments, the subject can benefit from administration of anutrient and the cargo moiety is a nutrient. Administration ofcochleates can be used to treat any of the diseases or disordersdescribed herein. In preferred embodiments, the compositions of theinvention are used to treat rhinosinusitis.

Administration of cochleates can be by a mucosal route, including oral,intranasal, intraocular, intrarectally, intravaginal, topical, buccaland intrapulmonary, or by a systemic route, including intravenous,intramuscular, intrathecal, subcutaneous, transdermal and intradermal.In a preferred embodiment the administration is intranasal. In anotherembodiment, the cochleate composition is delivered in the form of asolid, a capsule, a cachet, a pill, a tablet, a gelcap, a crystallinesubstance, a lozenge, a powder, a granule, a dragee, an electuary, apastille, a pessary, a tampon, a suppository, a patch, a gel, a paste,an ointment, a salve, a cream, a foam, a lotion, a partial liquid, anelixir, a mouth wash, a syrup, a spray, a nebulae, a mist, an atomizedvapor, an irrigant, an aerosol, a tincture, a wash, an inhalant, asolution or a suspension in an aqueous or non-aqueous liquid, and anoil-in-water and/or water-in-oil liquid emulsion. In a preferredembodiment, the cochleate composition is delivered in a form of a spray,a nebulae, a mist, an atomized vapor, an irrigant, an aerosol, a wash,and/or inhalant. In a preferred embodiment, the cochleate compositionincludes an antifungal drug. The antifungal drug can include any of theantifungal drugs discussed herein

In yet another aspect, the present invention involves an article ofmanufacture which includes packaging material and a lipid containedwithin the packaging material, wherein the packaging material comprisesa label or package insert indicating the use of the lipid for formingcochleates or cochleate compositions of the invention. In preferredembodiments, the article of manufacture can additionally includeinstructions or guidelines for the formation of cochleates or cochleatecompositions, a solvent, a phospholipid, a cargo moiety, a protonizedcargo moiety, a multivalent cation, a divalent metal cation, a controlcargo moiety, a chelating agent, and/or an aggregation inhibitor. Inparticularly preferred embodiments, one of the instructions involvesmixing a cargo moiety with a solvent and dripping it into a solution ofthe lipids.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is two fluorescent images of Rhodamine-labeled cochleatesincubated with splenocytes. These images demonstrate a transfer of lipidto the cell membrane, and indicate that a fusion event occurred betweenthe outer layer of the cochleate and the splenocyte cell membrane.

FIG. 2 illustrates an exemplary method of the present invention, whereindrug-liposomes are obtained by addition of a hydrophobic drug insolvent, optionally with an antioxidant, to a liposomal suspension.

FIG. 3 illustrates another aspect of the invention, wherein hydrosolubledrugs are encochleated.

FIG. 4 is a series of images of an Amphotericin B formulation having alipid to drug ratio of 1:1 at different stages in the formulation:liposomes, liposomes with AmB, cochleates, and cochleates after additionof EDTA.

FIGS. 5, 6 and 7 are each a series of images, before and after additionof EDTA, of AmB-cochleate formulations having a lipid to drug ratio of10:1, 2:1, and 1:1 ratio, respectively.

FIG. 8 is a graph of the size distribution of the liposomes aftervortexing and prior to filtration, after filtration with 0.45 μm filter,and after introducing DMSO/Amphotericin.

FIG. 9 is a graph of the size distribution of cochleate formulationshaving lipid to AmB ratios of 10:1, 2:1 and 1:1.

FIG. 10 is a graph of the survival data for C. albicans-infected miceuntreated (control), or dosed daily for 14 days with AmB/deoxycholate,or AmB-cochleates with a lipid to drug ratio of 2:1, 3:1, 4:1, or 5:1.

FIG. 11 is a chart of the average number of C. albicans cells/gram oftissue in the liver, kidney, and lungs of C. albicans-infected miceuntreated and dosed daily for 14 days with AmB/deoxycholate, orAmB-cochleates with a lipid to drug ratio of 2:1, 3:1, 4:1, or 5:1.

FIG. 12 is a graph of the number of colony forming units (CFU) for theC. albicans-infected macrophages dosed with varying concentrations ofAmB-cochleates with lipid to drug ratios of 2:1, 3:1, 4:1, and 5:1.

FIG. 13 is a series of images of the 5:1 AmB cochleates (top two panels)and the cochleates after addition of EDTA (bottom two panels).

FIG. 14 is a graph of the survival data for the C. albicans-infectedmice untreated or dosed daily for 14 days with AmB/deoxycholate(AmB/ID), or AmB-cochleates with a lipid to drug ratio of 5:1(dialysis), 2:1 (dialysis), 1:1 (dialysis), or 2:1 (wash).

FIG. 15 is a chart of the average number of C. albicans cells/gram oftissue in the liver, kidney, and lungs of C. albicans-infected miceuntreated (control), or dosed daily for 14 days with AmB/deoxycholate(AmB/D), or AmB-cochleates with lipid to drug ratios of 5:1 (dialysis),2:1 (dialysis), 1:1 (dialysis), or 2:1 (washing).

FIG. 16 is a graph depicting the number of colony forming units (CFUs)for C. albicans-infected macrophages dosed with varying concentrations(0.1, 0.01 and 0.001 μl/mg) of AmB-cochleate formulations having lipidto drug ratios of 5:1 (dialysis), 2:1 (dialysis), 1:1 (dialysis), and2:1 (washing), or AmB/deoxycholate (AmB/D).

FIG. 17 is a graph of the survival data for C. albicans-infected miceuntreated (control), or dosed daily for 14 days with AmB/deoxycholate orAmB-cochleates (CAMB) in suspension or lyophilized and formulated withor without methylcellulose (MC).

FIG. 18 is a chart of the average number of C. albicans cells/gram oftissue in the liver, kidney, and lungs of C. albicans-infected miceuntreated and dosed daily for 14 days with AmB/deoxycholate orAmB-cochleates (CAMB) in suspension or lyophilized and formulated withor without methylcellulose (MC).

FIG. 19 is a graph of the concentration of TY-cochleate preparationsversus free TY over time.

FIG. 20 is two graphs of the concentration of each impurity over timefor both the free TY and TY-cochleates studied in FIG. 21.

FIG. 21 is a graph comparing the cytotoxicity of TY-cochleates in aSKOV3 cell line.

FIG. 22 is an image of ZnTPP in solution (100% DMSO), andZnTPP-cochleates. The ZnTPP in solution was dark purple, and thecochleate formulation was only slightly colored (pink), indicating thatthe ZnTPP was successfully incorporated into the cochleates, which arewhite/yellowish.

FIG. 23 is a series of phase contrast images (left panels) andfluorescence images (right panels), of the ZnTPP-cochleates (top panels)and ZnTPP-liposomes (bottom panels) formed. These images indicate thatthe ZnTPP was successfully associated with the liposomes andsuccessfully encochleated.

FIG. 24 is a series of phase contrast images (left panels) andfluorescence images (right panels), of ZnTPP-cochleates (top panels) andZnTPP liposomes (bottom panels) formed without the presence of solvent.

FIG. 25 is a series of images of the SKOV3 cell culture with the ZnTPPcochleates at 1 hour and 24 hours.

FIG. 26 is a series of images of the SKOV3 cell culture with the freeZnTPP (in DMSO) at 1 hour and 24 hours.

FIG. 27 is a series of images of the SKOV3 cell culture with the emptycochleates (including DOPE-pyrene lipid) at 1 hour and 24 hours.

FIG. 28 is a series of images of the SKOV3 cell culture with theZnTPP-cochleates (including DOPE-pyrene lipid) at 1 hour and 24 hours.

FIG. 29 is a series of images illustrating the use of an exemplary kitof the invention, this is a model compound used as a control.

FIG. 30 is a schematic diagram of cochleate aggregation in the absenceof an aggregation inhibitor.

FIG. 31 is a schematic diagram of an exemplary method of makingcochleates of the invention by adding an aggregation inhibitor prior tocochleate formation.

FIG. 32 is a schematic diagram of an exemplary method of makingcochleates of the invention by adding an aggregation inhibitorsubsequent to cochleate formation.

FIG. 33A is two fluorescent images demonstrating the uptake of standardcochleates by cultured cells. FIG. 33B is two fluorescent imagesdemonstrating the uptake of cochleates of the invention by culturedcells.

FIGS. 34A and 34B are two graphs depicting the size distribution ofcochleates of the invention and standard cochleate aggregates.

FIGS. 35A-D are four fluorescent images of Rhodamine-labeled cochleatesdemonstrating the effect of formulating cochleates in the presence ofvarious aggregation inhibitors: half and half (FIG. 35A), whole milk(FIG. 35B), and fat-free milk (FIG. 35C). FIG. 35D is an image of acontrol composition of cochleates that do not include an aggregationinhibitor.

FIGS. 36A and 36B are two fluorescent images of Rhodamine-labeledcochleates demonstrating the disaggregation of cochleates upon additionof an aggregation inhibitor.

FIG. 37A is two images comparing acetaminophen cochleates with andwithout aggregation inhibitor (casein). FIG. 37B is two images comparingaspirin cochleates with and without an aggregation inhibitor (casein).

FIG. 38 is a graph comparing the in vivo efficiency of coated (small)and standard (large) aspirin cochleates at different concentrations andwith optional additive in reducing edema in rat paws injected withcarrageenan.

FIG. 39 is a graph showing the extent of ulceration and bleeding in ratstreated with free indomethacin, free aspirin, standard aspirincochleates, and aspirin cochleates with an aggregation inhibitor versusan untreated control.

FIG. 40 is a graph indicating that empty cochleates are immunologicallyinert in that they have no effect on the production of NO inmacrophages.

FIG. 41 is a graph comparing the efficacy of encochleated andunencochleated aspirin and acetaminophen cochleates in inhibiting NOformation.

FIG. 42 is an image of a cochleate prepared in the absence of calcium.

FIG. 43 is an image of a cochleate prepared in the presence of calcium.

FIG. 44 is an image of a cochleate prepared in the presence of calciumand subsequently treated with a molar excess of chelating agent (EDTA).

FIGS. 45A-D are images depicting vancomycin-lipid formulations. FIG. 45Adepicts vanco-liposomes. FIG. 45B depicts vanco-cochleates that includean aggregation inhibitor (casein). FIG. 45C depicts vanco-cochleateswithout an aggregation inhibitor. FIG. 45D depicts the cochleates ofFIG. 45C upon addition of a chelator (EDTA).

FIG. 46 is a graph summarizing efficacy data for free vancomycin, andvancomycin cochleates with and without casein on S. aureus at 3 hours.

FIG. 47 is a graph summarizing efficacy data for free vancomycin, andvancomycin cochleates with and without casein on S. aureus at 6 hours.

FIG. 48 are images depicting tobramycin-lipid formulations. FIG. 48Adepicts tobramycin-liposomes. FIG. 48B depicts tobramycin-cochleatesthat include an aggregation inhibitor (casein). FIG. 48C depictstobramycin-cochleates without an aggregation inhibitor. FIG. 48D depictsthe cochleate of FIG. 48C upon addition of a chelator (EDTA).

FIG. 49 is a graph summarizing efficacy data for free tobramycin andtobramycin cochleates with and without casein on P. aeruginosa alone andcultured in macrophages at 3 hours.

FIG. 50 is a graph summarizing efficacy data for free tobramycin, andtobramycin cochleates with and without casein on P. aeruginosa alone andcultured in macrophages at 6 hours.

FIG. 51 is a series of images of 10:1 soy PS:caspofungin cochleatesbefore (FIG. 51A) and after (FIG. 51B) addition of EDTA.

FIG. 52 is a series of images of 5:1 soy PS:caspofungin cochleatesbefore (FIG. 52A) and after (FIG. 52B) addition of EDTA.

FIGS. 53A and 53B are two graphs demonstrating the size distribution of10:1 soy PS:caspofungin (FIG. 53A) and 5:1 soy PS:caspofungin (FIG. 53B)cochleates.

FIG. 54 is a graph demonstrating the size distribution of 10:1 soyPS:caspofungin cochleates before and after addition of bovine serumalbumin and homogenization.

FIGS. 55A and 55B are two HPLC spectra depicting the contents of openedcaspofungin cochleates. In all formulations only caspofungin is evident.

FIG. 56 is a graph depicting the stability of caspofungin cochleatesformulated in water, in saline and in saline with additional calcium.

FIG. 57 is a series of images depicting caspofungin cochleates atvarying pH. Caspofungin cochleates appear most stable at a pH range ofabout 4-6.

FIG. 58 is a series of images depicting Amphotericin B cochleates with a5:1 lipid:drug ratio containing 0.2% parabens and varying amounts ofmethylcellulose.

FIG. 59 is a graph depicting particle size distribution of AmphotericinB cochleates with a 5:1 lipid:drug ratio containing varying amounts ofmethylcellulose or 0.2% parabens.

FIG. 60 is an HPLC spectrum depicting the contents of openedAmphotericin B cochleates formed using an exemplary method of theinvention. Only Amphotericin B is present in the cochleate.

FIG. 61 is a graph depicting the number of colony forming units (CFUs)for C. albicans-infected macrophages dosed with varying concentrations(5, 1, 0.1, 0.01 and 0.001 μl/mg) of AmB-cochleate formulations havinglipid to drug ratios of 5:1 with and without 0.3% methylcellulose insuspension and lyophilized to form a powder.

FIG. 62 is a graph depicting the particle size distribution ofamphotericin B cochleates formulated in a large batch (>5 L) with aninset of the image of the amphotericin B cochleates.

FIG. 63 is a graph depicting the particle size distribution ofamphotericin B cochleates formulated in a large batch (>5 L) withadditional rabbit serum albumin and passed through a homogenizer 2times. The inset is an image of the amphotericin B cochleates afterhomogenization.

FIG. 64 is a graph depicting the particle size distribution ofamphotericin B cochleates formulated in a large batch (>5 L) withadditional rabbit serum albumin and passed through a homogenizer 7times. The inset is an image of the amphotericin B cochleates afterhomogenization.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery of anovel method for the formulation of cochleates and cochleatecompositions. These cochleates and cochleate compositions provide allthe advantages of conventional cochleates, but are more efficientlymade, from a cost and a time perspective. The method generally includesthe step of introducing a cargo moiety to a liposome in the presence ofa solvent such that the cargo moiety associates with the liposome.

Without wishing to be bound to any particular theory, it is believedthat the solvent facilitates association of the cargo moiety with theliposome, e.g., incorporation of a cargo moiety with the liposomalbilayer. For example, in some embodiments, a hydrophobic or amphipathiccargo moiety is dissolved in the solvent prior to addition to an aqueousliposomal suspension. When the solution is added to the liposomalsuspension, the solvent is miscible with the water which changes thepolarity and decreases the solubility of the cargo moiety in thesolution. The cargo moiety then associates, at least in part, with themore hydrophobic environment of the lipid bilayer. For example, thehydrophobic portion of an amphipathic molecule may associate-itself withthe lipid bilayer, leaving the remainder of the molecule to resideoutside the liposome.

In another embodiment, the cargo moiety is hydrosoluble and/orhydrophilic, and the solvent creates an environment such that the cargomoiety associates with the lipid bilayer (e.g., by ionic attraction tothe lipid and/or cation and/or total or partial migration into thebilayer). Additionally or alternatively, the solvent also may facilitatemembrane permeation of the cargo moiety (e.g., an alcohol may beemployed to enhance the permeability of the liposomal bilayer).

The invention also provides cochleates and cochleate compositions (e.g.,pharmaceutical compositions), prepared by the methods of the invention.

The present invention also provides novel cochleates and cochleatecompositions that include an aggregation inhibitor. These cochleates andcochleate compositions provide all the advantages of conventionalcochleates, and additionally provide a cochleate or cochleatecomposition having a stable mean particle size and distribution. Thecochleates and cochleate compositions can have, e.g., a small particlesize, e.g. a mean particle size of less that 600 nm, and/or a narrowparticle size range, e.g. less than about 700 nm. Moreover, thecomposition is stable and does not aggregate with the passage of time.

The invention also provides novel methods of formation of cochleatesthat allow the cochleates to be produced in any desired size range usinga variety of methods. These methods do not require such steps asproviding a two-phase system and/or particle size differentiation toobtain a cochleate composition having a mean particle size of less thana micrometer. The methods of the invention can be utilized with anyknown method of making cochleates.

The present invention also provides a composition for the safe andefficient delivery of cargo moieties in anhydrous cochleates. Theinvention is based, at least in part, on the discovery that protonizedcargo moieties can be precipitated with a negatively charged lipid and adivalent metal cation to form anhydrous, stable and safe compositionsfor delivery of the moiety. Moreover, protonized cargo moieties can beincluded in the cochleates at surprisingly high concentrations, ifdesired.

The cochleates of the invention not only protect the cargo moiety fromthe host (e.g., from decomposition by proteolytic enzymes in thedigestive tract), but also protect the host from the cargo moiety (e.g.,preventing damage to vital organs caused by toxic levels of certaincargo moieties). In addition, the cochleates of the invention allow forefficient delivery of the cargo moiety across the digestive tract and tocells, e.g., by fusion and/or cellular uptake. Thus, a lower dosage ofcargo moiety can be administered to generate the same beneficial resultsas compared to conventional preparations, while minimizing the incidenceof toxic side effects and/or buildup of cargo moiety in the digestivetract

The methods of the invention are superior to those employingconventional liposomal preparations. By way of example,liposome-encapsulated tobramycin has resulted in a low bactericidalactivity in vitro. In contrast, anhydrous tobramycin cochleatepreparations of the present invention facilitate oral delivery withlower serum levels of drug, thereby lowering the toxicity. In addition,the anhydrous tobramycin cochleates of the invention may be absorbed viathe gastrointestinal tract and delivered directly to the site ofinfection. Moreover, once anhydrous tobramycin cochleates are within thesystemic circulation, they can be efficiently accumulated by phagocytesresulting in intracellular delivery of the drug to infected cells.

The invention also provides methods for forming cochleates that includecontacting a protonized cargo moiety, a negatively charged lipid and adivalent metal cation.

The invention further provides methods of using the cochleates of theinvention, including methods of administration. Finally, the inventionprovides methods of use, including therapeutic use, and kits directed tothe manufacture and use of the cochleates and cochleate compositions ofthe invention.

In order to more clearly and concisely describe the subject matter ofthe claims, the following definitions are intended to provide guidanceas to the meaning of specific terms used in the following writtendescription, examples and appended claims.

The term “cargo moiety,” as used herein, refers to a moiety to beencochleated, and generally does not refer to the lipid and ion employedto precipitate the cochleate. Cargo moieties include any compoundshaving a property of biological interest, e.g., ones that have a role inthe life processes of a living organism. A cargo moiety may be organicor inorganic, a monomer or a polymer, endogenous to a host organism ornot, naturally occurring or synthesized in vitro and the like.

As used herein, the terms “cochleate,” “lipid precipitate” and“precipitate” are used interchangeably to refer to a lipid precipitatecomponent that generally includes alternating cationic and lipid bilayersheets with little or no internal aqueous space, typically stackedand/or rolled up, wherein the cationic sheet is comprised of one or moremultivalent cations. Additionally, the term “encochleated” meansassociated with the cochleate structure.

The term “protonized cargo moiety” refers to a protonizable cargo moietythat has been protonized. “Protonizable” refers to the ability to gainone or more protons. The protonizable cargo moiety can be weakly basic,and can be protonized by acidification or addition of a proton.Additionally or alternatively, the protonizable cargo moiety can beneutral or weakly acidic and can be protonized in the same manner. Thus,the protonizable cargo moiety can be an anionic or a neutral cargomoiety, which is rendered cationic by protonization, or the protonizablecargo moiety can be cationic, and be rendered more cationic uponprotonization. The cargo moiety can also be provided protonized.Optionally, the protonized state can be induced, e.g., by acidificationor other methods, as described herein. Protonization renders the cargomoiety cationic or increases the valency of a cargo moiety that isalready cationic, e.g., from monovalent to divalent or trivalent.

The term “protonization,” as used herein, refers to the process ofincreasing the valency of a cargo moiety. “Protonized” refers to a cargomoiety that has undergone protonization. Thus, valency can be increased,e.g. from 0 to 1, from 1 to 2, from 2 to 3, from 3 to 4, or anycombination thereof, e.g., from 0 to 3. Any method to increase valency,e.g., increasing pH, can be used to protonize a cargo moiety.

The term “weakly basic cargo moiety” refers to cargo moieties that, atneutral pH, have the ability to accept protons. That is, weakly-basiccargo moieties are capable of being rendered cationic or more cationicby protonization. As such, weakly basic cargo moieties can be anionic orneutral, and be rendered cationic by protonization. Alternatively,weakly basic cargo moieties can be cationic, and can be rendered morecationic, i.e., polycationic, by protonization.

The term “weakly acidic cargo moiety” refers to cargo moieties that, atneutral pH, have the ability to give up protons. “Protonizable weaklyacidic cargo moieties,” due to their weak acidity, have the ability toaccept protons at decreased pH.

“Aminioglycoconjugates,” as used herein, refer to compounds that includean amino sugar or carbohydrate covalently linked with another moiety.Exemplary subgroups of aminoglycoconjugates include, but are not limitedto, aminoglycoproteins, aminoglycosides, glycosaminoglycans, andaminoglycopeptides.

“Aminoglycopeptides” are compounds that include an amino sugar orcarbohydrate covalently linked to one or more peptides, includingsynthetic or chemically modified derivatives. Aminoglycopeptidesinclude, but are not limited to vancomycin, teicoplanin, bleomycin,peptidolglycan, ristocetin, sialoglycoproteins, orienticin, avaporcin,helevecardin, galacardin, and actinoidin. Derivatives of these compoundsalso are included, e.g., those provided by reductive alkylation ofreactive amines. See, Sundram et al., J. Org. Chem. 60:1102-03 (1995).U.S. Pat. Nos. 4,639,433, 4,643,987, and 4,698,327, teach N-alkyl andN-acyl derivatives of vancomycin. European Patent Nos. 435 503A1 and 667353 A1, described reductive alkylations of a variety ofaminoglycopeptides including vancomycin and orienticin A.

“Aminoglycosides” are compounds that include at least two amino sugarslinked by glycoside bonds to a streptidine or a 2-deoxystreptamine ortheir analogs. Analogs are meant to include aminoglycosides modified,e.g., to increase resistance to enzyme cleavage. Such derivatives (e.g.,amikacin, a semisynthetic derivative of kanamycin), are necessary fortreatment of individuals or populations that have built up resistance toother aminoglycosides, and all such derivatives developed presently orin the future are aminoglycosides that fall within the scope of thepresent invention. Analogs also are meant to include the structurallyrelated aminocyclitols (e.g., spectinomycin). Aminoglycosides include,but are not limited to, gentamicin, netilmicin, tobramycin, amikacin,kanamycin A, kanamycin B, neomycin, paromomycin, neamine, streptomycin,dihydrostreptomycin, apramycin, ribostamycin, and spectinomycin.Aminoglycosides can optionally be grouped as streptomycins (e.g.,streptomycin and dihydrostreptomycin), kanamycins (e.g., kanamycin,amikacin, tobramycin), gentamicins (e.g., gentamicin and netilmicin),and neomycins. Apramycin and specinomycin are aminoglycosides typicallyused by veterinarians to treat non-human animals.

“Echinocandins” are large lipopeptide molecules, which are active asantifungal agents. Echinocandins act by inhibiting glucan synthesis viainhibition of 1,3-beta-D-glucan synthase. This interferes with thesynthesis of chitin, an important cell-wall component, and results infungal cell lysis. These drugs have fungicidal activity against a vastspecies of fungi, e.g., Candida and Aspergillus. Examples ofechinocandins include, but are not limited to, caspofungin, echinocandinB, aculeacin A, micafungin, anidulafungin, cilofungin, and pneumocandin.Any antifungal molecule with an echinocandin core structure is meant tobe included.

As used herein, the term “peptide” refers to a compound containing twoor more amino acids, such as a protein. The term “nucleotide” refers toone or more purine or pyrimidine molecules attached to a backbone. Thebackbone can be a sugar-phosphate backbone, or a modified backbone,e.g., a morpholino backbone. The terms “protonized peptide” and“protonized nucleotide” are meant to include any peptide or nucleotidethat can be rendered divalent or polyvalent.

“Carbohydrates” include any carbohydrate including those that includeone or more monosaccharides, disaccharides, oligosaccharides orpolysaccharides, and their derivatives.

Cochleates

Cochleate delivery vehicles represent a unique technology platformsuitable for the oral and systemic administration of a wide variety ofmolecules with important therapeutic biological activities, includingdrugs, genes, and vaccine antigens. Miller et al., J Exp Med176:1739-1744 (1992); Gould-Fogerite and Mannino, J. Liposome Res6(2):357-79 (1996); Mannino and Gould-Fogerite, New Generation Vaccines,ch. 18, pp 229-39 (Marcel Dekker, New York, N.Y., Myron M. Levine, Ed.2nd ed. 1997); Gould-Fogerite et al., Advanced Drug Delivery Reviews32(3):273-387 (1998); Gould-Fogerite and Mannino, Methods in MolecularMedicine, Vaccine Adjuvants: Preparation Methods and Research Protocolspp 179-196 (Humana Press, Totowa 1999); Gould-Fogerite et al., JLiposome Research 10(4): 339-358 (2000); U.S. Pat. No. 5,834,015;Gould-Fogerite et al., Gene 84:429-438 (1989); Zarif et al., J. LiposomeResearch-60(4), 523-538 (2000); Zarif et al., Antimicrobials Agents andChemotherapy 44(6):1463-1469 (2000); Santangelo et al., AntimicrobialsAgents and Chemotherapy 44(9):2356-2360 (2000); Parker et al., Methodsin Enzymology: Antisense Technology, Part B, 314: 411-29 (M. IanPhillips, Ed., 1999).

Cochleate formulation technology is particularly applicable tomacromolecules as well as small molecule drugs that are hydrophobic,positively charged, negatively charged and/or possess poor oralbioavailability. Proof-of-principle studies for cochleate mediated oraldelivery of macromolecules as well as small molecule drugs is beingcarried out in appropriate animal models with well established,clinically important drugs which currently can only be effectivelydelivered by injection (e.g., antifungal agents such as amphotericin B).

The cochleate structure provides protection from degradation forassociated “encochleated” moieties. Divalent cation concentrations invivo in serum and mucosal secretions are such that the cochleatestructure is maintained. Hence, the majority of cochleate-associatedmolecules are present in the inner layers of a primarily solid,non-aqueous, stable, impermeable structure. Since the cochleatestructure includes a series of solid layers, components within theinterior of the cochleate structure remain substantially intact, eventhough the outer layers of the cochleate may be exposed to harshenvironmental conditions or enzymes. In an exemplary method of cochleateformation, liposomes, which include negatively charged lipids associatedwith a cargo moiety, are exposed to a cation, e.g., calcium, thatinteracts with the liposomes to displace water and condense the lipid.The cation/lipid sheets “roll-up” and/or stack against each other tominimize contact with water, which provides an environment for theencochleated molecule that is substantially free of water. Thisstructure provides protection to encochleated molecules from digestionin the stomach.

The cochleate interior is primarily free of water and resistant topenetration by oxygen. Oxygen and water are primarily responsible forthe decomposition and degradation of cargo moieties (e.g., drugs andnutrients), which leads to reduced shelf-life. Accordingly,encochleation also imparts extensive shelf-life stability. For example,for DNA vaccine-cochleates, the encochleation efficiency, the percentageof supercoiled versus relaxed plasmid, and immunogenicity are equivalentto fresh preparations for more than one year.

With respect to storage, cochleates can be stored in cation-containingbuffer, or lyophilized or otherwise converted to a powder, and stored atroom temperature. If desired, the cochleates also can be reconstitutedwith liquid prior to administration. Cochleate preparations have beenshown to be stable for more than two years at 4° C. in acation-containing buffer, and at least one year as a lyophilized powderat room temperature.

As used herein, the term “multivalent cation” refers to a divalentcation or higher valency cation, or any compound that has at least twopositive charges, including mineral cations such as calcium, barium,zinc, iron and magnesium and other elements capable of forming ions orother structures having multiple positive charges capable of chelatingand bridging negatively charged lipids. Additionally or alternatively,the multivalent cation can include other multivalent cationic compounds,e.g., cationic or protonized cargo moieties. The term “divalent metalcation,” as used herein, refers to a metal having two positive charges.

The lipid employed in the present invention preferably includes one ormore negatively charged lipids. As used herein, the term “negativelycharged lipid” includes lipids having a head group bearing a formalnegative charge in aqueous solution at an acidic, basic or physiologicalpH, and also includes lipids having a zwitterionic head group.

The cochleates of the invention also can include non-negatively chargedlipids (e.g., positive and/or neutral lipids). Preferably, a majority ofthe lipid is negatively charged. In one embodiment, the lipid is amixture of lipids, comprising at least 75% negatively charged lipid. Inanother embodiment, the lipid includes at least 85% negatively chargedlipid. In other embodiments, the lipid includes at least 90%, 95% oreven 99% negatively charged lipid. All ranges and values between 60% and100% negatively charged lipid are meant to be encompassed herein.

The negatively charged lipid can include soy-based lipids. Preferably,the lipid includes phospholipids, such as soy-based phospholipids. Thenegatively charged lipid can include phosphotidyl serine (PS),dioleoylphosphatidylserine (DOPS), phosphatidic acid (PA),phosphatidylinositol (PI), and/or phosphatidyl glycerol (PG) and or amixture of one or more of these lipids with other lipids. Additionallyor alternatively, the lipid can include phosphatidylcholine (PC),phosphatidylethanolamine (PE), diphosphotidylglycerol (DPG), dioleoylphosphatidic acid (DOPA), distearoyl phosphatidylserine (DSPS),dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylglycerol(DPPG) and the like.

The lipids can be natural or synthetic. For example, the lipid caninclude esterified fatty acid acyl chains, or organic chains attached bynon-ester linkages such as ether linkages (as described in U.S. Pat. No.5,956,159), disulfide linkages, and their analogs.

In one embodiment the lipid chains are from about 6 to about 26 carbonatoms, and the lipid chains can be saturated or unsaturated. Fatty acyllipid chains useful in the present invention include, but are notlimited to, n-tetradecanoic, n-hexadecanoic acid, n-octadecanoic acid,n-eicosanoic acid, n-docosanoic acid, n-tetracosanoic acid,n-hexacosanoic acid, cis-9-hexadecenoic acid, cis-9-octadecenoic acid,cis,cis-9,12-octadecedienoic acid, all-cis-9,12,15-octadecetrienoicacid, all-cis-5,8,11,14-eicosatetraenoic acid,all-cis-4,7,10,13,16,19-docosahexaenoic acid, 2,4,6,8-tetramethyldecanoic acid, and lactobacillic acid, and the like.

The cochleates of the invention can further include additional compoundsknown to be used in lipid preparations, e.g., cholesterol, and/orpegylated lipid. Pegylated lipid includes lipids covalently linked topolymers of polyethylene glycol (PEG). PEG's are conventionallyclassified by their molecular weight, thus PEG 6,000 MW, e.g., has amolecular weight of about 6000. Adding pegylated lipid generally willresult in an increase of the amount of compound (e.g., peptide,nucleotide, and nutrient) that can be incorporated into the cochleate.An exemplary pegylated lipid is dipalmitoylphosphatidylehtanolamine(DPPE) bearing PEG 5,000 MW.

Methods of Forming Cochleates

In one aspect, the invention provides methods for forming cochleates.Any known method can be used to form cochleates, including but notlimited to those described in U.S. Pat. Nos. 5,994,318 and 6,153,217,the entire disclosures of which are incorporated herein by thisreference.

In one embodiment, the method generally includes introducing a cargomoiety to a lipid in the presence of a solvent, adding an aqueoussolution to form liposomes, and precipitating to form a cochleate.

In a preferred embodiment, the method generally includes introducing acargo moiety to a liposome in the presence of a solvent such that thecargo moiety associates with the liposome, and precipitating theliposome to form a cargo moiety-cochleate.

The step of introducing a cargo moiety to a liposome in the presence ofa solvent can be achieved in a variety of ways, all of which areencompassed within the scope of the present invention. In oneembodiment, the cargo moiety is introduced by introducing a solution ofthe solvent and the cargo moiety to the liposome. Preferably, theliposome is in a liposomal suspension, preferably, an aqueous liposomalsuspension. In a preferred embodiment, the solution is introduced to theliposome by dropwise addition of the solution. In other embodiments, thesolution can be added by continuous flow or as a bolus. In addition thesolution may be introduced to dried lipid, with water added before,after or with the solution.

In another embodiment, the cargo moiety is introduced to the liposomeprior to or after the solvent. For example, the cargo moiety may beintroduced to a liposomal suspension that includes the solvent. Themixture can then be agitated, mixed, vortexed or the like to facilitateassociation of the cargo moiety with the liposome. The cargo moietyintroduced may be in a powder or a liquid form.

An antioxidant (e.g., Vitamin E) may also be employed in the methods ofthe present invention. It can be introduced with the cargo moiety orwith the liposome. Preferably, it is incorporated into the liposomalsuspension or a solution of the cargo moiety and solvent.

The liposome may be prepared by any known method of preparing liposomes.Thus, the liposomes may be prepared for example by solvent injection,lipid hydration, reverse evaporation, freeze drying by repeated freezingand thawing. The liposomes may be multilamellar (MLV) or unilamellar(ULV), including small unilamellar vesicles (SUV). The concentration oflipid in these liposomal solutions can be from about 0.1 mg/ml to 500mg/ml. Preferably, the concentration of lipid is from about 0.5 mg/ml toabout 50 mg/ml, more preferably from about 1 mg/ml to about 25 mg/ml.

The liposomes may be large unilamellar vesicles (LUV), stableplurilamellar vesicles (SPLV) or oligolamellar vesicles (OLV) prepared,e.g., by detergent removal using dialysis, column chromatography, biobeads SM-2, by reverse phase evaporation (REV), or by formation ofintermediate size unilamellar vesicles by high pressure extrusion.Methods in Biochemical Analysis, 33:337 (1988). Liposomes made by allthese and other methods known in the art can be used in practicing thisinvention.

In a preferred embodiment at least majority of the liposomes areunilamellar. The method can further include the step of filtering aliposomal suspension and/or mechanically extruding the suspensionthrough a small aperture that includes both MLV and ULV liposomes, suchthat a majority of the liposomes are ULV. In preferred embodiments, atleast 70%, 80%, 90% or 95% of the liposomes are ULV.

The method is not limited by the method of forming cochleates. Any knownmethod can be used to form cochleates from the liposomes of theinvention (i.e., the liposomes associated with the cargo moiety). In apreferred embodiment, the cochleate is formed by precipitation. Theliposome can be precipitated with a multivalent cation to form a cargomoiety-cochleate. The multivalent cation can consist entirely or consistessentially of a cationic metal, including, but not limited to calcium,magnesium, barium, zinc, and/or iron. Additionally or alternatively, themultivalent cation can include other multivalent cationic compounds. Asused herein, the term “multivalent” refers to ions having a valency ofat least 2, e.g., divalent, trivalent, etc.

Any suitable solvent can be employed in connection with the presentinvention. Solvents suitable for a given application can be readilyidentified by a person of skill in the art. Preferably, the solvent isan FDA acceptable solvent.

The solvent can be an organic solvent or an inorganic solvent. In oneembodiment, the solvent is a water miscible solvent. Suitable solventsinclude but are not limited to dimethylsulfoxide (DMSO), amethylpyrrolidone, N-methylpyrrolidone (NMP), acetonitrile, alcohols,e.g., ethanol (EtOH), dimethylformamide (DMF), tetrahydrofuran (THF),and combinations thereof. In general, the cargo moiety concentrationwithin the solvent is between about 0.01 mg/ml and 200 mg/ml.Preferably, the cargo moiety concentration is between about 0.05 mg/mland about 100 mg/ml, more preferably between about 0.1 mg/ml and 20mg/ml.

The solvent can optionally be removed, e.g., before the formation ofliposomes, at the liposome stage and/or after the cochleates are formed.Any known solvent removal method can be employed. For example, solventmay be removed from the liposomal suspension by tangential flow and/orfiltration and/or dialysis, or from the cochleates by washing,filtration, centrifugation, and/or dialysis. The cochleates can bewashed, e.g., with buffer or water, optimally with calcium or anothercation.

Utilizing the methods of the invention a wide range of lipid to cargomoiety ratios can be achieved. Different ratios can have varyingbiological activity. The amount of cargo moiety incorporated into thecochleates can be varied as desired. The optimal lipid:cargo moietyratio for a desired purpose can readily be determined without undueexperimentation. The cochleates can be administered to the targeted hostto ascertain the nature and tenor of the biologic response to theadministered cochleates. It is evident that the optimized ratio for anyone use may range from a high ratio to a low ratio to obtain maximalamount of cargo moiety in the cochleates. All ratios disclosed hereinare w/w, unless otherwise indicated. In one embodiment, the ratio oflipid to cargo moiety is between about 10,000:1 and 1000:1. Ratios inthis range may be suitable when it is desired to administer smallamounts of the moiety, (e.g., in the case of administration ofradioactive agents or highly active, rare or expensive molecules). Inanother embodiment, the ratio is between about 8,000:1 and 4,000:1,e.g., about 6,000:1. Such a ratio may be suitable, e.g., in deliveringporphyrins. In yet another embodiment, the ratio is between about5,000:1 and 50:1. In yet another embodiment, the ratio of the lipid tothe cargo moiety is between about 20:1 and about 0.5:1. In anotherembodiment, the ratio of the lipid to the cargo moiety is between about1:1 and about 10:1. Such a ratio may be suitable, e.g., for delivery ofan antifungal agent. In yet another embodiment, the ratio of lipid tothe cargo moiety is about 2:1, about 3:1, or between about 1.5:1 and3.5:1. All individual values and ranges between about 0.25:1 and about40,000:1 are within the scope of the invention. Further values also arewithin the scope of the invention. The cochleate formulations also canbe prepared both with and without targeting molecules (e.g., fusogenicmolecules, such as Sendai virus envelope polypeptides), to targetspecific cells and/or tissues.

In some embodiments, the cargo moiety is hydrophobic. In others, it isamphipathic. In still others, it is hydrophilic and/or hydrosoluble.Exemplary cargo moieties are disclosed below.

In preferred embodiments, hydrophobic cargo moiety cochleates (e.g.,beta-carotene cochleates) are formed by introducing a hydrophobic cargomoiety to a liposome in the presence of a solvent such that thehydrophobic cargo moiety associates with the liposome, and precipitatingthe liposome to form a hydrophobic cargo moiety-cochleate. Inparticularly preferred embodiments, the loading of hydrophobic cargomoiety in the cochleate is considerably higher than the loading observedwhen cochleates are formed using conventional methods, i.e., thosedescribed in U.S. Pat. No. 5,994,318.

Formation of the cochleates of the invention in the above methodsinvolves crystallization of multivalent cation with negatively chargedlipids. It is evident, therefore, that all of the parameters that governcrystallization, e.g., temperature, lipid concentration, multivalentcation concentrations, rate of cation addition, pH and rate of mixing,can be utilized to regulate cochleate formation. In certain embodiments,ionic conditions can be created or adjusted to affect the efficiency ofthe association and/or the encochleation of the cargo moiety. Forexample, increasing the salt concentration in a liposomal suspension canrender the environment less hospitable to a hydrophobic or amphipathiccargo moiety, thereby increasing liposome and cochleate loadingefficiency. Ionic conditions can also affect the ultimate structure ofthe cochleate generated. High loads of a cargo moiety can also affectthe highly ordered structure observed in cochleates formed, e.g.,exclusively from calcium and PS. Additionally or alternatively, pHconditions can be created or adjusted to affect the loading andstructure of the resulting cochleates. Such variations can readily bemanipulated by the skilled practitioner using no more than the instantspecification and routine experimentation. In addition, because acochleate is highly thermodynamically stable, once a cochleateformulation method is developed for a given product, the end product canbe made predictably and reliably.

Accordingly, in another aspect, the present invention provides methodsof making anhydrous cochleates with protonized cargo moieties. Themethod generally includes the step of contacting a negatively chargedlipid, a protonized cargo moiety and a divalent metal cation. Withoutwishing to be bound by any particular theory, it is believed that thenegatively charged lipid forms an ionic interaction with the cationicprotonized-cargo moiety. The divalent metal cation then precipitates thelipid and protonized cargo moiety to form an anhydrous cochleate.

In a preferred embodiment, the protonized cargo moiety is introduced tothe negatively charged lipid. A divalent metal cation is then added tothe lipid-protonized cargo moiety mixture in order to form anhydrouscochleates. The divalent metal cation can be, e.g., calcium, barium,etc. In particularly preferred embodiments, the divalent metal cation iscapable of inducing the formation of an anhydrous cochleate. In otherparticularly preferred embodiments, the divalent metal cation iscalcium.

In one embodiment, liposomes are formed that include negatively chargedlipid using known methods, and the protonized cargo moiety is addedprior to, during or after formation of the liposomal suspension.Alternatively, the protonized cargo moiety is introduced to a preformedliposomal suspension, e.g., as a solid or in an aqueous or organicsolution.

The method can further include the step of protonizing the cargo moietyprior to or during the formation of the cochleate, e.g., byacidification. Any known method of acidification can be employed. Forexample, a weakly basic cargo moiety can be protonized with acidicaqueous buffer. A buffer is chosen based upon the pK_(a) of the cargomoiety. A cargo moiety with a lower pK_(a) would necessitate a bufferwith a lower pH range than that of a cargo moiety with a higher pK_(a).Thus, for caspofungin, with pK_(a), values of 5.1, 8.7, 9.7 and 10.7, abuffer with a pH range of between 4.5 and 5.5 would be sufficient tomaintain its multivalency. Buffers with a pH range suitable foracidification can readily be identified by the skilled practitionerbased upon the cargo moiety being protonized. Suitable buffers includelow molecular weight buffers having an acidic pK_(a), e.g., amino acidsand TES. Acidic buffers are known in the art, and identification of avariety of acidic buffers would require no more than routineexperimentation by one of ordinary skill in the art. Alternatively, theweakly basic cargo moiety can be protonized by slow addition of an acid,e.g., hydrochloric acid, to an aqueous solution of lipid and weaklybasic cargo moiety.

In still other embodiments, the protonizable cargo moiety has more thanone pK_(a) value. In preferred embodiments, the pH is lowered to belowthe highest pK_(a) value. In other preferred embodiments, pH is loweredto below the second highest pK_(a) value. In other preferredembodiments, the pH is lowered to below the third, fourth, fifth or evensixth highest pK_(a) value. In yet other preferred embodiments, the pHis lowered to below the lowest pK_(a) value.

Optionally, the cargo moiety can be protonized in the lipid-cargo moietymixture, e.g., by lowering the pH or introducing the cargo moiety to asuspension of lipids at a low pH. Because of its cationic nature, theprotonized cargo moiety tends to associate with the negatively chargedsurface of the liposome bilayers.

In yet other embodiments, the cargo moieties can be protonized prior toincorporation into the cochleates. For example, they can be obtained orpurchased protonized from the manufacturer. Additionally oralternatively, they can be protonized, isolated as a protonized cargomoiety, and subsequently incorporated into a cochleate at a suitable pH.A suitable pH is a pH that allows the cargo moiety to remain protonized,and can be readily determined by the skilled artisan.

In other embodiments, the pH of the resultant anhydrous cochleates insolution can be adjusted using, e.g., acid. Without wishing to be boundby any particular theory, it is believed that this would help tomaintain the protonized cargo moiety within the cochleate structure.

In another aspect, the present invention generally is directed tomethods of making cochleates that include an aggregation inhibitor. Theaggregation inhibitor can be introduced prior to, during or afterformation of cochleates. That is, the aggregation inhibitor can be addedto the lipid-cargo moiety solution, to the liposomal solution or to theprecipitated cochleate. For example, in one embodiment, the aggregationinhibitor is introduced to a liposomal suspension from which cochleateswill subsequently be formed (e.g., by addition of cation or dialysis).That is, the aggregation inhibitor may be introduced prior to formationof liposomes, e.g., it may be added to dried lipid prior to suspensionor added directly to a liposomal suspension, before of after addition ofa cargo moiety. In such embodiments, the cochleates may be initiallyformed in the desired size range and aggregation thereafter prevented bythe presence of the aggregation inhibitor.

In other embodiments, the methods of the invention can include the stepof introducing an aggregation inhibitor to a cochleate composition. Forexample, the method can further include forming cochleates (prior tointroducing the aggregation inhibitor). The method can include providingcochleates already formed, e.g., cochleates obtained from a supplier.The method can further include the step of disaggregating cochleates byadding an aggregation inhibitor to aggregated cochleates.

In still other embodiments, aggregated cochleates may be disaggregatedusing alternative disaggregation methods, e.g., homogenization, and anaggregation inhibitor can be introduced in order to preventreaggregation.

In yet another embodiment, the aggregation inhibitor can be introducedduring the formation of the cochleate, e.g., it can be added with thecation or during dialysis.

In a preferred embodiment, the aggregation inhibitor is added in anamount suitable for modulating the resulting cochleate to the desiredsize.

The method can include forming cochleates with any or all of theoptional ingredients disclosed herein. For example, the cochleates caninclude additional cationic compounds, protonized cargo moieties,non-negative lipids, and/or aggregation inhibitors.

Any of the methods described herein can be utilized to produce anywherefrom about 1 mg to about 500 g of cochleates in one batch. A smallerbatch may be preferred in a laboratory setting where characterization ofcochleates is desired. On the other hand, larger batches may bepreferred in a manufacturing setting where mass production is desired.Preferably, larger batches are at least 50 g, and more preferably atleast 75 g.

FIG. 2 illustrates an exemplary method of the present invention, whereindrug-liposomes are obtained by addition of a hydrophobic drug in solvent(e.g., DMSO, DMF, THF, EtOH), optionally with an antioxidant (e.g.,Vitamin E), to a liposomal suspension. A liposomal suspension isprepared by vortexing lipid (e.g., soyPS) and water and filtered,however, other methods of obtaining liposomal suspensions can beemployed in the methods of the present invention. Cochleates areprecipitated out by the addition of calcium (e.g., calcium chloride),and subsequently can be washed (e.g., with calcium containing buffers)to remove any residual solvent, if desired. Alternatively, residualsolvent can be removed by other methods, e.g., dialysis.

FIG. 3 illustrates another aspect of the invention, wherein hydrosolubledrugs are encochleated. In this method, a hydrosoluble drug is addeddirectly to liposomes and subsequently precipitated. The liposomes areprepared by adding lipid (e.g., dry Soy PS powder) to water, but couldbe prepared or provided by any other known means.

Aggregation Inhibitors

In some preferred embodiments, the cochleates of the present inventioncan optionally include one or more aggregation inhibitors. The term“aggregation inhibitor,” as used herein, refers to an agent thatinhibits aggregation of cochleates. The aggregation inhibitor typicallyis present at least on the surface of the cochleate, and may only bepresent on the surface of the cochleate (e.g., when the aggregationinhibitor is introduced after cochleate formation). Aggregationinhibitors can be added before, after, or during cochleate formation.The type and/or amount of aggregation inhibitor can be adjusted toobtain a desired cochleate size and/or distribution. Additionally oralternatively, aggregation inhibitor(s) can be used to stabilizecochleate size and/or size distribution such that aggregation ofcochleates is minimized or eliminated.

Such compositions are advantageous for several reasons including thatsmaller cochleates can allow for greater uptake by cells and rapidefficacy. Such a composition is suitable, e.g., when it is desired torapidly and effectively deliver a cargo moiety (e.g., an antifungal orantibacterial agent against a fungal or bacterial infection). Moreover,particle size can have a targeting affect in that some cells may take upparticles of a certain size more or less effectively. Size may alsoaffect the manner in which cochleates interact with a cell (e.g., fusionevents or uptake).

Aggregation inhibitors work in part by modifying the surfacecharacteristics of the cochleates such that aggregation is inhibited.Aggregation can be inhibited, for example, by steric bulk around thecochleate, which inhibits aggregation and/or changes the nature of thecochleate structure, e.g., a change in the surface hydrophobicity and/orsurface charge.

The terms “coat,” “coated,” “coating,” and the like, unless otherwiseindicated, refer to an agent (e.g. an aggregation inhibitor) present atleast on the outer surfaces of a cochleate. Such agents may beassociated with the bilayer by incorporation of at least part of theagent into the bilayer, and/or may be otherwise associated, e.g., byionic attraction to the cation or hydrophobic or ionic attraction to thelipid.

As discussed herein, cochleates can be formed by the calcium inducedrestructuring and fusion of lipid, e.g., phospholipid such asphosphatidylserine (PS). Due to the hydrophobic nature of the surfacesof cochleates in aqueous, calcium containing solutions, cochleatesformed without the aggregation inhibitors of the invention can aggregateand form larger masses, e.g., needle-like structures in aspirincochleates (FIG. 37A, right panel). It has been discovered thatrestricting and/or inhibiting the interaction of liposomes that cancoalesce into cochleates at the time of cation addition limits the sizeof the resultant cochleate crystal, and prevents aggregation into largerparticles. The addition of an aggregation inhibitor (e.g., casein) toliposomes prior to the addition of calcium results in stablenon-aggregated nanocochleate structures (FIG. 37A, left panel).

The type and/or amount of aggregation inhibitor used can also determinethe size of resulting cochleate. The presence of an aggregationinhibitor in differing concentrations also allows regulation ofcochleate size distribution.

It also has been discovered that addition of one or more aggregationinhibitors after formation of cochleates also inhibits and even reversesaggregation. For example, it is shown in FIG. 35 that the addition ofhalf and half, whole milk, and fat free milk to Rhodamine-PE cochleatesinhibits aggregation. It can also be noted that the milk products withmore fat content (milk and half and half) inhibited aggregation morethan the fat free milk, which has less fat content. Additionally, theaddition of an aggregation inhibitor (milk) to aggregated cochleates hasbeen demonstrated to disaggregate the cochleates as depicted in FIG. 36.

Suitable aggregation inhibitors that can be employed in accordance withthe present invention, include but are not limited to at least one ofthe following: casein, κ-casein, milk, albumin, serum albumin, bovineserum albumin, rabbit serum albumin, methylcellulose, ethylcellulose,propylcellulose, hydroxycellulose, hydroxymethyl cellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose,polyvinyl pyrrolidone, carboxymethyl cellulose, carboxyethyl cellulose,pullulan, polyvinyl alcohol, sodium alginate, polyethylene glycol,polyethylene oxide, xanthan gum, tragacanth gum, guar gum, acacia gum,arabic gum, polyacrylic acid, methylmethacrylate copolymer, carboxyvinylpolymer, amylose, high amylose starch, hydroxypropylated high amylosestarch, dextrin, pectin, chitin, chitosan, levan, elsinan, collagen,gelatin, zein, gluten, carrageenan, carnauba wax, shellac, latexpolymers, milk protein isolate, soy protein isolate, whey proteinisolate and mixtures thereof.

A preferred aggregation inhibitor is casein. Casein is a highlyphosphorylated, calcium binding protein. Without wishing to be bound toany particular theory, it is believed that calcium mediates aninteraction between negatively charged lipid (e.g., PS) and casein,thereby changing the surface properties of cochleates such thataggregation is inhibited. Another preferred aggregation inhibitor ismilk and other milk products such as Half and Half, cream etc. Preferredmilk products also contain casein. Another preferred aggregationinhibitor is an excipient, e.g., methylcellulose. Other preferredaggregation inhibitors include albumin, serum albumin, bovine serumalbumin and rabbit serum albumin.

More than one aggregation inhibitor may be employed in the compositionsof the invention. For example, both milk and methylcellulose may be usedas an aggregation inhibitor.

In one embodiment, the cochleate compositions of the invention includebetween about 10% and about 0.1% aggregation inhibitor. Preferably, theaggregation inhibitor comprises about 1% of the cochleate composition.

In another embodiment, the cochleate compositions of the inventioninclude an aggregation inhibitor to lipid ratio of between about 0.1:1to about 4:1 by weight.

Preferably, the aggregation inhibitor to lipid ratio is about 1:1. Aperson of ordinary skill in the art will readily be able to determinethe amount of aggregation inhibitor needed to form cochleates of thedesired size with no more than routine experimentation.

Cochleate Size and Distribution

The formation of cochleates can be envisioned as a crystallization eventthat spontaneously occurs upon the interaction of charged lipids andoppositely charged multivalent cations. Modulating of the size ofcochleate crystals formed, however, has prior to the present inventionproved difficult.

In aqueous suspension, plain cochleates generally aggregate and uponlong term storage form larger masses which can be several microns insize. Because of the association of the calcium with the lipid headgroup, the surfaces of cochleates have a hydrophobic character. Whensuspended in aqueous buffer, cochleate aggregation is a consequence ofhydrophobic interactions, minimizing the amount of surface area exposedto water. FIG. 30 is a schematic model of cochleate aggregation inaqueous solution.

It has been discovered that aggregation can be inhibited and evenreversed, and individual cochleate particles can be stabilized bychanging the surface properties of the cochleates and thereby inhibitingcochleate-cochleate interaction. Aggregation can be inhibited byincluding in the liposome suspension a material that preventsliposome-liposome interaction at the time of calcium addition andthereafter. Alternatively, the aggregation inhibitor can be added afterformation of cochleates. Additionally, the amount of aggregationinhibitor can be varied, thus allowing modulation of the size of thecochleates.

FIG. 31 is a schematic model of cochleates coated in proteins to reducethe amount of cochleate aggregation to near zero. As demonstrated ingreater detail below, the resulting cochleates are surprisingly small.Particle size analysis demonstrates that these formulations are stablenanocochleates. Additional experiments, presented below in the Examples,have extended these observations providing the conceptual basis for thedevelopment of protocols for the preparation of stabilized nanocochleateformulations of defined size.

FIG. 32 is a schematic diagram of an exemplary method of makingcochleates of the invention by adding an aggregation inhibitorsubsequent to cochleate formation. Accordingly, in one aspect, theinvention provides a cochleate composition comprising a plurality ofcochleates and an aggregation inhibitor. In a preferred embodiment, theaggregation inhibitor comprises a coating on the cochleates. Such a“coating” can be formed by addition of the aggregation inhibitor afterformation of cochleates. The amount of aggregation inhibitor employedand the point at which the aggregation inhibitor is added can be used tocontrol the particle sizes of the cochleates.

Accordingly, the present invention provides a cochleate compositioncomprising a plurality of cochleates and an aggregation inhibitor havinga desired particle size distribution, and methods of making the same. Asdemonstrated herein, the amount of aggregation inhibitor and/or time ofaddition can be varied to modulate and/or stabilize the size and/or sizedistribution of a cochleate composition.

In one embodiment, the aggregation inhibitor can be employed to achievecochleates that are significantly smaller and have narrower particlesize distributions than compositions without aggregation inhibitors asdemonstrated, e.g., in FIG. 34. Such compositions are advantageous forseveral reasons including that they can allow for greater uptake bycells (see e.g., FIG. 33), and rapid efficacy (see e.g., FIGS. 46, 47,49 and 50). Such a composition is suitable, e.g., when it is desired torapidly and effectively deliver a cargo moiety (e.g., an antifungal orantibacterial agent against a fungal or bacterial infection). Moreover,cochleate size can have a targeting affect in that some cells may takeup particles of a certain size more or less effectively. Size may alsoaffect the manner in which cochleates interact with a cell (e.g., fusionevents or uptake).

In another embodiment, the aggregation inhibitor can be employed in anamount to achieve cochleate-compositions having a particle sizerelatively larger than that which can be achieved without or with otheraggregation inhibitors (e.g., if more and/or a different aggregationinhibitor used). Such a composition can be useful, e.g., when delayeduptake and/or release of the cargo molecule is desired, or when targetedcells or organs more effectively take up cochleates in the relativelylarger size range. Such compositions also may have sustained activity(relative to smaller cochleate compositions) because it can take longerfor the cargo moiety to be released from a larger cochleate, e.g., ifmultiple fusion events are required.

In yet another embodiment, the amount and/or types of aggregationinhibitor can be chosen to manufacture a cochleate composition that hasa wide particle size distribution such that the cargo moiety is releasedover a period of time because smaller cochleates are rapidly taken upinitially followed by take up or fusion events with increasingly largercochleates. In addition, size may not only affect what type of cellstake up the cochleate, but also how the cochleates interact with certaincells, e.g., size may effect whether a cochleate is taken up by a cellor undergoes one or more fusion events with a cell.

Moreover, in yet further embodiments, several compositions can becombined for desired release profiles, e.g., a pulsed released, orcombined release. For example, a rapid release nanocochleate compositioncan be mixed with a delayed-release larger size or even standardcochleate composition, such that an immediate and a delayed release areboth realized. In an exemplary case, both small and large antibioticcochleates are administered in order to treat a subject with a highinitial dose (small cochleates) and to maintain enough antibiotic in theserum to be effective against remaining bacteria (large cochleates). Inaddition, the cochleate compositions may have different cargo moieties,e.g., a stomach protecting medication can be formulated withnanocochleates for initial release (or a large distribution for longterm release), and one or more non-steroidal anti-inflammatory drugs canbe formulated with larger cochleates (NSAID) for release after thestomach protecting medication is released.

An aggregation inhibitor also can be employed to stabilize particle sizeand particle size distribution. For example, it can be used to “lock-in”the cochleate size and distribution of standard cochleates and/orcochleates having an aggregation inhibitor. While the cochleates of theinvention typically are stable over long periods of time, standardcochleates (cochleates formed without aggregation inhibitors) can tendto aggregate over time. Thus, standard cochleates can be reduced in sizeand/or stabilized by addition to such aggregation inhibitors, e.g.,addition of methylcellulose after cochleate formation. FIG. 54 shows thedecrease in size of caspofungin cochleates which have been homogenizedand treated with bovine serum albumin.

Cochleates formed in the presence of aggregation inhibitors do notaggregate. Accordingly, such compositions are advantageous for severalreasons including, e.g., greater uptake by cells, and increasedefficacy. Cochleate compositions of the invention preferably have a meandiameter less than about 5, 4, 3, 2, or 1 micrometer. Preferably, thecochleate compositions have a mean diameter less than about 900 nm, 800nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm. Allindividual values between these values (880, 435, 350), are meant to beincluded and are within the scope of this invention. In anotherembodiment, cochleate compositions of the invention include cochleatepopulations having a mean diameter about equal to or greater than about1 micrometer, e.g., 2, 3, 4, 5, 10, 50, or 100 micrometers. Allindividual values and ranges within these ranges are meant to beincluded and are within the scope of this invention.

Preferably, the size distribution is narrow relative to that observed instandard cochleates (cochleates formed without aggregation inhibitors).As demonstrated, e.g. in FIG. 34, the size distribution of cochleatecompositions with aggregation inhibitors is significantly improvedrelative to that observed in standard cochleate compositions.Preferably, the cochleates have a size distribution of less than about30, 20, 10, 5, 3 or 1 μm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400nm, 300 nm, 200 nm, or 100 nm. All individual values between thesevalues (550 nm, 420 nm, 475 nm, etc.), are meant to be included and arewithin the scope of this invention. Such compositions are particularlydesirable where uptake by macrophages is desired. It can readily beappreciated that particle size can be adjusted to a size suitable foruptake by desired organs or cells and/or unsuitable for uptake by organsor cells. In another embodiment, a wider size distribution of cochleatesis employed, e.g., about 10, 20, 50, 100, 200 . . . 500 micrometers. Allindividual values within these ranges are meant to be included and arewithin the scope of this invention. Such compositions can be useful forlong term release of cargo moieties.

Additionally, as discussed above, the invention contemplates combinationof cochleate populations with one or more cargo moieties, one or moresize distributions, and one or more mean diameter, to achieve a desiredrelease pattern, e.g., pulsed release, delayed release and/or timedrelease of different cargo-moieties.

Cargo Moieties

The cochleates of the present invention are preferably associated or“loaded” with a cargo moiety. A “cargo moiety” is a moiety to beencochleated, and generally does not refer to the lipid and ion employedto precipitate the cochleate. Cargo moieties include any compoundshaving a property of biological interest, e.g., ones that have a role inthe life processes of a living organism. A cargo moiety may be organicor inorganic, a monomer or a polymer, endogenous to a host organism ornot, naturally occurring or synthesized in vitro and the like.

Thus, examples include vitamins, minerals, nutrients, micronutrients,amino acids, toxins, microbicides, microbistats, co-factors, enzymes,polypeptides, polypeptide aggregates, polynucleotides, lipids,carbohydrates, nucleotides, starches, pigments, fatty acids, saturatedfatty acids, monounsaturated fatty acids, polyunsaturated fatty acids,flavorings, essential oils, extracts, hormones, cytokines, viruses,organelles, steroids and other multi-ring structures, saccharides,metals, metabolic poisons, antigens, imaging agents, porphyrins,tetrapyrrolic pigments, drugs and the like.

The cargo moiety can be a diagnostic agent, such as an imaging agent.Imaging agents include nuclear agents and fluorescent probes, e.g.,porphyrins. Porphyrins include tetrapyrrolic agents or pigments. Onesuch tetrapyrrolic agent is Zinc Tetra-Phenyl Porphyrin (ZnTPP), whichis a hydrophobic, fluorescent molecule that has high absorption in thevisible spectrum (dark purple).

The polynucleotide can be one that is expressed to yield a biologicallyactive polypeptide or polynucleotide. Thus, the polypeptide may serve asan immunogen or, for example, have enzymatic activity. Thepolynucleotide may have catalytic activity, for example, be a ribosome,or may serve as an inhibitor of transcription or translation, e.g., asmall interfering RNA (siRNA) or an antisense molecule. Thepolynucleotide can be an antisense molecule including modified antisensemolecule, such as a morpholino antisense molecule. The polynucleotidecan be modified, e.g., it can be synthesized to have a morpholinobackbone. If expressed, the polynucleotide preferably includes thenecessary regulatory elements, such as a promoter, as known in the art.A specific example of a polypeptide is insulin.

The cargo moiety can be an organic molecule that is hydrophobic inaqueous media. The cargo moiety can also be a water-soluble monovalentor polyvalent cationic molecule, anionic, or net neutral atphysiological pH.

The drug can be, but is not limited to, a protein, a small peptide, abioactive polynucleotide, an antibiotic, an antiviral, an anesthetic,antipsychotic, an anti-infectious, an antifungal, an anticancer, animmunosuppressant, an immunostimulant, a steroidal anti-inflammatory, anon-steroidal anti-inflammatory, an antioxidant, an antidepressant whichcan be synthetically or naturally derived, a substance which supports orenhances mental function or inhibits mental deterioration, ananticonvulsant, an HIV protease inhibitor, a non-nucleophilic reversetranscriptase inhibitor, a cytokine, a tranquilizer, a mucolytic agent,a dilator, a vasoconstrictor, a decongestant, a leukotriene inhibitor,an anti-cholinergic, an anti-histamine, a cholesterol lipid metabolismmodulating agent or a vasodilatory agent. The drug can also be any overthe counter (non-prescription) medication.

An antifungal drug can be a polyene macrolide, tetraene macrolide,pentaenic macrolide, fluorinated pyrimidine, imidazole, azole, triazole,halogenated phenolic ether, thiocarbamate, allylamine, sterol inhibitor,and an agent that interpolates fungal cell wall components.

Nonsteroidal anti-inflammatory drugs (NSAIDS) are typically used totreat inflammation, muscle strains, and high fever. NSAIDS function byinhibiting. cyclooxygenase-1 (COX1) and cyclooxygenase-2 (COX2). COX1enzymes are responsible for protecting the lining of the stomach andCOX2 enzymes are responsible for the production of prostaglandins, whichare important in the inflammatory process. Unfortunately, commerciallyavailable preparations of NSAIDS are active against both COX1 and COX2,and therefore have unwanted side effects such as ulcers, upset stomachor nausea.

Examples of suitable drugs include Amphotericin B, acyclovir,adriamycin, carbamazepine, ivermectin, melphalen, nifedipine,indomethacin, curcumin, aspirin, ibuprofen, naproxen, acetaminophen,rofecoxib, diclofenac, ketoprofen, meloxicam, nabumetone, estrogens,testosterones, steroids, phenyloin, ergotamines, cannabinoids,rapamycin, propanadid, propofol, alphadione, echinomycin, miconazole,miconazole nitrate, ketoconazole, itraconazole, fluconazole,griseofulvin, clotrimazole, econazole, terconazole, butoconazole,oxiconazole, sulconazole, saperconazole, voriconazole, ciclopiroxolamine, haloprogin, tolnaftate, naftifine, terbinafine hydrochloride,morpholines, flucytosine, natamycin, butenafine, undecylenic acid,Whitefield's ointment, propionic acid, and caprylic acid, clioquinol,selenium sulfide, teniposide, hexamethylmelamine, taxol, taxotere,18-hydroxydeoxycorticosterone, prednisolone, dexamethasone, cortisone,hydrocortisone, piroxicam, diazepam, verapamil, vancomycin, tobramycin,teicoplanin, bleomycin, peptidolglycan, ristocetin, sialoglycoproteins,orienticin, avaporcin, helevecardin, galacardin, actinoidin, gentamycin,netilmicin, amikacin, kanamycin A, kanamycin B, neomycin, paromomycin,neamine, streptomycin, dihydrostreptomycin, apramycin, ribostamycin,spectinomycin, caspofungin, echinocandin B, aculeacin A, micafungin,anidulafungin, cilofungin, pneumocandin, geldanamycin, nystatin,rifampin, tyrphostin, a glucan synthesis inhibitor, vitamin A acid,mesalamine, risedronate, nitrofurantoin, dantrolene, etidronate,nicotine, amitriptyline, clomipramine, citalopram, dothiepin, doxepin,fluoxetine, imipramine, lofepramine, mirtazapine, nortriptyline,paroxetine, reboxetine, sertraline, trazodone, venlafaxine, dopamine,St. John's wort, phosphatidylserine, phosphatidic acid, amastatin,antipain, bestatin, benzamidine, chymostatin, 3,4-dichloroisocoumarin,elastatinal, leupeptin, pepstatin, 1,10-phenanthroline, phosphoramidon,ethosuximide, ethotoin, felbamate, fosphenytoin, lamotrigine,levitiracetam, mephenyloin, methsuximide, oxcatbazepine, phenobarbital,phensuximide, primidone, topirimate, trimethadione, zonisamide,saquinavir, ritonavir, indinavir, nelfinavir, and amprenavir.

Tyrphostin and geldanamycin (GA) target the oncoprotein/oncogene erb B2,which is overexpressed on a variety of tumor cells, and this high levelof expression is functionally related to transformation.

GA is a hydrophobic small molecule drug that has been shown to haveactivity in vitro against cancer cell lines. It inhibits ErbB2expression by destabilizing chaperone proteins. GA has beentraditionally dissolved in DMSO for in vitro and in vivo testing. Invivo, it has anti-tumor activity, but has significant hepatotoxicity.Tyrphostin AG-825 is a tyrosine kinase inhibitor that has activityagainst cancer cell lines over-expressing erb B2. It inhibits itsactivity, and therefore cellular proliferation, but not erb B2expression.

The drug can be a polypeptide such as cyclosporin, Angiotensin I, II andIII, enkephalins and their analogs, ACTH, anti-inflammatory peptides I,II, III, bradykinin, calcitonin, b-endorphin, dinorphin, leucokinin,leutinizing hormone releasing hormone (LHRH), insulin, neurokinins,somatostatin, substance P, thyroid releasing hormone (TRH) andvasopressin.

The drug can be an antigen, but is not limited to a protein antigen. Theantigen can also be a carbohydrate or DNA. Examples of antigenicproteins include membrane proteins, carbohydrates, envelopeglycoproteins from viruses, animal cell proteins, plant cell proteins,bacterial proteins, and parasitic proteins.

The antigen can be extracted from the source particle, cell, tissue, ororganism by known methods. Biological activity of the antigen need notbe maintained. However, in some instances (e.g., where a protein hasmembrane fusion or ligand binding activity or a complex conformationwhich is recognized by the immune system), it is desirable to maintainthe biological activity. In these instances, an extraction buffercontaining a detergent which does not destroy the biological activity ofthe membrane protein is employed. Suitable detergents include ionicdetergents such as cholate salts, deoxycholate salts and the like orheterogeneous polyoxyethylene detergents such as Tween, BRIG or Triton.

Utilization of this method allows reconstitution of antigens into theliposomes with retention of biological activities, and efficientassociation with the cochleates. The method may also be employed withoutsonication, extreme pH, temperature, or pressure all of which may havean adverse effect upon efficient reconstitution of the antigen in abiologically active form.

Suitable nutrients include, but are not limited to lycopene,micronutrients such as phytochemicals or zoochemicals, vitamins,minerals, fatty acids, amino acids, fish oils, fish oil extracts,saccharides, herbal products and essential oils and flavor agents.Specific examples include Vitamins A, B, B1, B2, B3, B12, B6, B-complex,C, D, E, and K, vitamin precursors, caroteniods, and beta-carotene,resveratrol, biotin, choline, inositol, gingko, lutein, zeaxanthine,quercetin, silibinin, perillyl alcohol, genistein, sulfurophane, andessential fatty acids, including eicosapentaenoic acid (EPA), gamma-3,omega-3, gamma-6 and omega-6 fatty acids, herbs, spices, and iron.Minerals include, but are not limited to boron, chromium, colloidalminerals, colloidal silver, copper, manganese, potassium, selenium,vanadium, vanadyl sulfate, calcium, magnesium, barium, iron and zinc.

As used herein, “micronutrient” is a nutrient that the body must obtainfrom outside sources. Generally micronutrients are essential to the bodyin small amounts.

The cargo moiety can be a saccharide or sweetener, e.g., saccharine,isomalt, maltodextrin, aspartame, glucose, maltose, dextrose, fructoseand sucrose. Flavor agents include oils, essential oils, or extracts,including but not limited to oils and extracts of cinnamon, vanilla,almond, peppermint, spearmint, chamomile, geranium, ginger, grapefruit,hyssop, jasmine, lavender, lemon, lemongrass, marjoram, lime, nutmeg,orange, rosemary, sage, rose, thyme, anise, basil, black pepper, tea ortea extracts, an herb, a citrus, a spice or a seed.

In some preferred embodiments, the cargo moiety can be a protonizedcargo moiety. In one embodiment, the cargo moiety is a protonized weaklybasic cargo moiety. The pharmacokinetics of weakly basic cargo moieties(e.g., vancomycin and tobramycin), conventionally has been dominated bytheir poor solubility in lipids such as milk. Because of this poorsolubility and the lack of water in the cochleates, it was surprisingthat weakly basic cargo moieties could be incorporated into cochleatesat the concentrations achieved in the present invention. It has beendiscovered, however, that protonized weakly basic cargo moieties can beincorporated into anhydrous cochleates. Protonized neutral cargomoieties can similarly be precipitated with negatively charged lipid,provided that acidification renders them cationic. Additionally, cargomoieties suitable for use in accordance with the present invention caninclude protonized weakly acidic cargo moieties or protonized amphotericcargo moieties. Weakly acidic cargo moieties or amphoteric cargomoieties may or may not include an initial positive charge. Such cargomoieties would also be rendered cationic by protonization. Protonizablecargo moieties can be negatively charged, positively charged, unchargedor zwitterionic. The invention is particularly advantageous in thepreparation of protonized water-soluble cargo moieties.

In one embodiment, the protonized cargo moiety is monovalent. In otherembodiments, the protonized cargo moiety is multivalent, e.g., divalent,trivalent, etc. In certain embodiments, a higher valency may bepreferable due to the size and/or conformation of the cargo moiety.

Moreover, because the protonized cargo moieties are cationic, hydrouscochleates can be a made without additional cation (e.g., a metalcation, such as calcium). For example, vancomycin-cochleates have beenmade without cation, as described below. Anhydrous cochleates made withdivalent metal cation, e.g., Ca²⁺, are preferred and are active againstStaph. A. infection in vitro.

In one embodiment, the protonized cargo moiety is a multivalent cation(i.e., polycationic). The protonization or acidification can render anon-cationic moiety cationic or increase the valency of a cationicmoiety. The protonized cargo moiety can optionally be isolated andcharacterized prior to formulation into a cochleate. Alternatively, thecargo moiety can be obtained or purchased protonized (e.g., vancomycinhydrochloride or caspofungin acetate).

In one embodiment, the protonized cargo moiety is a protonized peptide,such as a protonized protein.

In another embodiment, the protonized cargo moiety is a protonizednucleotide. The protonized nucleotide can be, but is not limited to aprotonized DNA, a protonized RNA, a protonized morpholino, a protonizedsiRNA molecule, a protonized ribozyme, a protonized antisense molecule,or a protonized plasmid.

In a preferred embodiment, the cargo moiety is a drug, including, butnot limited to, an aminoglyconjugate, e.g., an aminoglycoside or anaminoglycopeptide. Preferably the aminoglycoconjugate is weakly basic.

In a particularly preferred embodiment, the aminoglycoconjugate is oneor more of the following: vancomycin, teicoplanin, bleomycin,peptidolglycan, ristocetin, sialoglycoproteins, orienticin, avaporcin,helevecardin, galacardin, actinoidin, gentamycin, netilmicin tobramycin,amikacin, kanamycin A, kanamycin B, neomycin, paromomycin, neamine,streptomycin, dihydrostreptomycin, apramycin, ribostamycin, andspectinomycin.

In another preferred embodiment, the cargo moiety is an echinocandin. Ina particularly preferred embodiment, the echinocandin is one or more ofthe following: caspofungin, echinocandin B, aculeacin A, micafungin,anidulafungin, cilofungin, and pneumocandin.

The cochleates of the invention can be prepared with a wide range ofcargo moiety to lipid ratios. By way of example, the ratio of cargomoiety to lipid can be between about 20,000:1 and about 0.5:1 by weight.In one embodiment the ratio is about 1:1 by weight. In others the ratiois about 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 50:1, 100:1, 200:1, or 400:1 byweight. All individual ranges and values between 20,000:1 and 0.5:1 areencompassed by the invention.

The cochleates of the present invention can optionally include one ormore additional cargo moieties. The additional cargo moiety can be asecond protonized cargo moiety or any other cargo moiety.

Additional pharmacologically active agents may be delivered incombination with the primary active agents, e.g., the cochleates of thisinvention. In one embodiment, such agents include, but are not limitedto agents that reduce the risk of atherosclerotic events and/orcomplications thereof. Such agents include, but are not limited tobeta-blockers, beta blockers and thiazide diuretic combinations, HMG CoAreductase inhibitors, statins, aspirin, ace inhibitors, ace receptorinhibitors (ARBs), and the like.

Suitable beta blockers include, but are not limited to cardioselective(selective beta 1 blockers), e.g., acebutolol (e.g., Sectral™), atenolol(e.g., Tenormin™), betaxolol (e.g., Kerlone™), bisoprolol (e.g.,Zebeta™), metoprolol (e.g., Lopressor™), and the like. Suitablenon-selective blockers (block beta 1 and beta 2 equally) include, butare not limited to carteolol (e.g., Cartrol™), nadolol (e.g., Corgard™),penbutolol (e.g., Levatol™), pindolol (e.g., Visken™), propranolol(e.g., Inderal™), timolol (e.g., Blockadren™), labetalol (e.g.,Normodyne™, Trandate™), and the like.

Suitable beta blocker thiazide diuretic combinations include, but arenot limited to Lopressor HCT, ZIAC, Tenoretic, Corzide, Timolide,Inderal LA 40/25, Inderide, Normozide, and the like.

Suitable statins include, but are not limited to pravastatin (e.g.,Pravachol™), simvastatin (e.g., Zocor™), lovastatin (e.g., Mevacor™),and the like.

Suitable ace inhibitors include, but are not limited to captopril (e.g.,Capoten™), benazepril (e.g., Lotensin™), enalapril (e.g., Vasotec™),fosinopril (e.g., Monopril™), lisinopril (e.g., Prinivil™ or Zestril™),quinapril (e.g., Accupril™), ramipril (e.g., Altace™), imidapril,perindopril erbumine (e.g., Aceon™), trandolapril (e.g., Mavik™), andthe like. Suitable ARBS (Ace Receptor Blockers) include but are notlimited to losartan (e.g., Cozaar™), irbesartan (e.g., Avapro™),candesartan (e.g., Atacand™), valsartan (e.g., Diovan™), and the like.

Suitable HMG CoA reductase inhibitors that are useful in accordance withthe methods and compositions of the invention are statin molecules.These include: Lovastatin (e.g., Mevacor™), Pravastatin (e.g.,Pravachol™), Simvastatin (e.g., Zocor™), Fluvastatin (e.g., Lescol™),Atorvastatin (e.g., Lipitor™), or Cerivastatin (e.g., Baycol™).

Other agents that may be administered in conjunction with the cochleatesof the invention for treatment of atherosclerotic events and/orcomplications thereof are phytosterols, phytostanols and theirderivatives and isomers; soy protein; soluble fibers, e.g. beta-glucanfrom, for example, oat and psyllium, nuts, rice bran oil, each of whichis particularly suitable for use in food, dietary supplements and foodadditive compositions. Phytosterols may be solid (e.g., powder,granules) or liquid (e.g., oil) form.

It will be obvious to a person of skill in the art that the choice ofthe agent for treatment of atherosclerotic events and/or complicationsthereof depends on the intended delivery vehicle (e.g., food,supplement, pharmaceutical) and the mode of administration.

The cargo moiety can additionally be bound to a cochleate component orto a hydrophobic tail. In one embodiment, the cargo moiety is bound tothe lipid cochleate component or the hydrophobic tail with a digestible,reducible, or otherwise reversible linker. The cargo moiety can be boundin a reversible manner e.g., with a reducible or digestible linker) or alinker susceptible to target conditions (e.g., pH, temperature,ultrasonic energy and the like). This is particularly useful as thelinker can be chosen such that it is readily digestible, e.g., by anenzyme, in the body generally or even in a target structure. Thus, e.g.,a linker can be chosen such that it is degraded by an enzyme in theplasma, interstitial fluids, in a cell (e.g. a macrophage) or in anendosome, such that the protonized cargo moiety becomes detached andavailable in unbound form in these structures. In another embodiment,the reversible linker can be an electrostatic or other bond that isbroken by a change in pH, e.g., in an organ or other structure in whichthe cochleate experiences a pH gradient. In another embodiment, thelinker is reversed by a change in temperature, e.g., by exposure to bodytemperature.

In one embodiment, the cargo moiety is bound by an electrostatic,hydrophobic, covalent, or ionic interaction with a lipid component suchas a hydrophobic tail. In a preferred embodiment, the cargo moiety isbound to a component of the bilayer of the cochleate, e.g., aphospholipid or other lipid. Covalently binding the cargo moiety to thelipid by cross-linking can be accomplished by known methods. In oneembodiment, the covalent bond is reversible so that the cargo moiety canbe detached from the lipid component or hydrophobic tail under suitableconditions. For example, a cargo moiety can be attached to aphospholipid via a linker that can be cleaved by an enzyme endogenous toa target tissue, organ, or structure (e.g., a plasma protein,interstitial protein, an endosome or the intracellular milieu), suchthat the cargo moiety is delivered to the target tissue, organ or otherstructure. In alternative embodiments the cargo moiety can be attachedby any other means, for example, by electrostatic interactions and/orhydrophobic interactions.

The cargo moiety can be associated with the lipid component orhydrophobic tail in any of the methods described herein. For example, inone embodiment, the cargo moiety is associated with the lipid component,such that the cargo moiety dissociates with the lipid component uponcontact with a target environment. The cargo moiety can be bound to acomponent of the cochleate with any of the linkers described herein,e.g., a linker that is reducible, or otherwise reversible or digestibleby an enzyme, protein, or molecule endogenous to the target environment.The enzyme can be an extracellular, intracellular or endosomal enzymeendogenous to the subject. In another embodiment, the cargo moietycomponent is electrostatically associated with the lipid component anddissociates with the cochleate upon contact with a pH gradient in a cellor organ of the subject.

Delivery of Cargo Moieties

Many naturally occurring membrane fusion events involve the interactionof calcium with negatively charged phospholipids (e.g. PS andphosphatidylglycerol). Calcium-induced perturbations of membranescontaining negatively charged lipids, and the subsequent membrane fusionevents, are important mechanisms in many natural membrane fusionprocesses. Therefore, cochleates can be envisioned as membrane fusionintermediates.

Phase/fluorescent and fluorescent images of Rhodamine-labeled cochleatesincubated with splenocytes were captured and are shown in FIG. 1. Theseimages indicate that a fusion event occurs between the outer layer ofthe cochleate and the cell membrane, resulting in the delivery ofencochleated material into the cytoplasm of the target cell. As thecalcium rich, highly ordered membrane of a cochleate first comes intoclose approximation to a natural membrane, a perturbation and reorderingof the cell membrane is induced, resulting in a fusion event between theouter layer of the cochleate and the cell membrane. This fusion resultsin the delivery of a small amount of the encochleated material into thecytoplasm of the target cell. The cochleate can then break free of thecell and be available for another fusion event, either with the same oranother cell.

Additionally or alternatively, particularly with active phagocyticcells, cochleates may be taken up by endocytosis and fuse from withinthe endocytic vesicle. Cochleates made with trace amounts of fluorescentlipids have been shown to bind and gradually transfer lipids to theplasma membrane and interior membranes of white blood cells in vitro.FIG. 33, for example, demonstrates the uptake of cochleates bymacrophages.

Cochleates are useful for the delivery of a cargo moiety to culturedcells, tissues or organisms by a variety of administration routes. Theterm “delivery,” as used herein, refers to any means of bringing ortransporting a cargo moiety to a host, a food item, a formulation, apharmaceutical composition, or any other system, wherein the cargomoiety maintains at least a portion of its activity. For example, theuse of cochleates to deliver protein or peptide molecules as vaccineshas been disclosed in U.S. Pat. No. 5,840,707, issued Nov. 24, 1998.Similarly, polypeptide-cochleates are effective immunogens whenadministered to animals by intraperitoneal and intramuscular routes ofimmunization (G. Goodman-Snitkoff, et al., J. Immunol., Vol. 147, p. 410(1991); M. D. Miller, et al., J. Exp. Med., Vol. 176, p. 1739 (1992)).Further, cochleates are effective delivery vehicles for encapsulatedproteins and/or DNA to animals and to cells in culture. For example,reconstituted Sendai or influenza virus glycoproteins are efficientlydelivered in encochleated form (Mannino and Gould-Fogerite,Biotechniques 6(1):682-90 (1988); Gould-Fogerite et al., Gene 84:429(1989); Miller et al., J. Exp. Med. 176:1739 (1992)).

The cochleates can be coadministered with a further agent. The secondagent can be delivered in the same cochleate preparation, in a separatecochleate preparation mixed with the cochleate preparation of theinvention, separately in another form (e.g., capsules or pills), or in acarrier with the cochleate preparation. The cochleates can furtherinclude one or more additional cargo moieties, such as other drugs,peptides, nucleotides (e.g., DNA and RNA), antigens, nutrients, flavorsand/or proteins.

The cochleates of the invention also can include a reporter molecule foruse in in vitro diagnostic assays, which can be a fluorophore,radiolabel or imaging agent. The cochleates can include molecules thatdirect binding of the cochleate to a specific cellular target, orpromotes selective entry into a particular cell type.

One advantage of the cochleates of the present invention is thestability of the composition. Cochleates can be administered by anyroute, e.g., mucosal or systemic, without concern. Cochleates can beadministered orally or by instillation without concern, as well as bythe more traditional routes, such as oral, intranasal, intraoculate,intrarectal, intravaginal, intrapulmonary, topical, subcutaneous,intradermal, intramuscular, intravenous, transdermal, systemic,intrathecal (into CSF), and the like. Direct application to mucosalsurfaces is an attractive delivery means made possible with cochleates.Delivery can be effected by, e.g., a nasal spray or nasal bath orirrigation.

Another advantage of the present invention is the ability to modulatecochleate size. Modulation of the size of cochleates and cochleatecompositions changes the manner in which the cargo moiety is taken up bycells. For example, in general, small cochleates are taken up quicklyand efficiently into cells, whereas larger cochleates are taken up moreslowly, but tend to retain efficacy for a longer period of time. Also,in some cases small cochleates are more effective than large cochleatesin certain cells, while in other cells large cochleates are moreeffective than small cochleates.

Cochleates and cochleate compositions can also be administered to humansand non-human animals, such as dog, cats, and farm animals, in food orbeverage preparations. Such compositions can be introduced to the foodor beverage compositions by the manufacturer (e.g., to supplement foodwith nutrients), or by the consumer (e.g., where the cochleatecomposition is sold separately as a food additive). For example,nutrients and/or flavorings may be incorporated into dog or cat food,particularly where such nutrient and/or flavoring is fragile andnormally decomposes or loses activity when exposed to oxygen and/orwater. Cochleates may be added at any stage into the preparation of dogor cat food, as the cochleates are stable under extreme pressure andtemperature conditions.

Another advantage of cochleates and cochleate compositions of thepresent invention is their ability to reduce a number of unwanted sideeffects. A number of drugs currently on the market causegastrointestinal distress and often high circulating blood levels leadto toxicity in a number of vital organs. The ingestion of, e.g., aspirinmay result in epigastric distress, nausea, and vomiting. Aspirin mayalso cause gastric ulceration; exacerbation of peptic ulcer symptoms,gastrointestinal hemorrhage, and erosive gastritis have all beenreported in patients on high-dose therapy but also may occur even whenlow doses are administered. In high doses, aspirin can also causehepatic injury. Aspirin can cause retention of salt and water as well asan acute reduction of renal function in patients with congestive heartfailure or renal disease. Although long-term use of aspirin alone rarelyis associated with nephrotoxicity, the prolonged and excessive ingestionof aspirin in combination with other compounds can produce papillarynecrosis and interstitial nephritis. Although acetaminophen is usuallywell tolerated, skin rash (generally erythematous or urticarial) andother allergic reactions occur occasionally. Occasionally, the rash canbe more serious and may be accompanied by drug fever and mucosallesions. In other examples, the use of acetaminophen has been associatedwith neutropenia, thrombocytopenia, and pancytopenia. The most seriousadverse effect of acute overdosage of acetaminophen is a dose-dependent,potentially fatal hepatic necrosis. Renal tubular necrosis andhypoglycemic coma also may occur.

Another advantage of the present invention is that the cochleates can beformulated for uptake by particular cells or organs. Conventionally,high levels of drugs are often administered intravenously to obtainmoderate levels at the sites of infection in order to combatopportunistic infections. This can cause undesirable side effects, forexample, in the case of vancomycin, macular skin rashes, anaphylaxis,phlebitis and pain at the site of intravenous injection, chills, rash,and fever may occur. Also, rapid intravenous infusion may cause avariety of symptoms, including erythematous or urticarial reactions,flushing, tachycardia, and hypotension, generally non-permanent auditoryimpairment, ototoxicity associated with excessively high concentrationsof the drug in plasma and less commonly, nephrotoxicity. By employingthe cochleates of the present invention, toxicity levels can be loweredby decreasing the free drug in the circulating blood. Additionally, thecargo moiety can be delivered directly to the site of infection, whichcan lower or eliminate the incidence of gastrointestinal distress.

Aminoglycosides are very poorly absorbed from the gastrointestinaltract. Less than 1% of the dose typically is absorbed following eitheroral or rectal administration. Also, inadequate concentrations ofaminoglycosides are found in cerebrospinal fluid. Additionally, thedrugs are not inactivated in the intestine, and are excreted relativelyrapidly by the normal kidney, i.e., they are eliminated quantitativelyin the feces. Long-term oral or rectal administration, however, mayresult in accumulation of aminoglycosides to toxic concentrations inpatients with renal impairment. Instillation of these drugs into bodycavities with serosal surfaces may result in rapid absorption andunexpected toxicity, i.e., neuromuscular blockade. Similarly,intoxication may occur when aminoglycosides are applied topically forlong periods to large wounds, burns, or cutaneous ulcers, particularlyif there is renal insufficiency.

Moreover, due to their polar nature, aminoglycosides largely areexcluded from most cells, from the central nervous system, and from theeye. Concentrations of conventionally administered aminoglycosides insecretions and tissues are low. High concentrations, however, are foundin the renal cortex and in the endolymph and perilymph of the inner ear;this is thought to contribute to the nephrotoxicity and ototoxicitycaused by these drugs. Although they are widely used agents, serioustoxicity is a major limitation to the usefulness of the aminoglycosides.

Both vestibular and auditory dysfunction can follow the administrationof any of the aminoglycosides. Studies of both animals and human beingshave documented progressive accumulation of these drugs in the perilymphand endolymph of the inner ear. Accumulation occurs predominantly whenplasma concentrations are high. Diffusion back into the bloodstream isslow; the half-lives of the aminoglycosides are five to six times longerin the otic fluids than in plasma. Ototoxicity is more likely to occurin patients with persistently elevated concentrations of drug in plasma.However, even a single dose of tobramycin has been reported to produceslight temporary cochlear dysfunction during periods when theconcentration in plasma is at its peak. The relationship of thisobservation to permanent loss of hearing is not known.

Approximately 8% to 26% of patients who receive an aminoglycoside formore than several days develop renal impairment, which is almost alwaysreversible. The toxicity results from accumulation and retention ofaminoglycoside in the proximal tubular cells. The initial manifestationof damage at this site is excretion of the enzymes of the renal tubularbrush border. Several variables have been found to influencenephrotoxicity from aminoglycosides, including total amount of drugadministered and duration of therapy. Constant concentrations of drug inplasma above a critical level, which is manifested by elevated troughserum concentrations, correlate with toxicity in human beings.Aminoglycosides have the potential to produce reversible andirreversible vestibular, cochlear, and renal toxicity. These sideeffects complicate the use of these compounds and make their properadministration difficult.

Accordingly, the cochleates of the present invention can be employed toavoid harmful side effects of drugs caused by their high concentrationor presence in organs such as the kidneys, stomach or liver.

Echinocandins are a relatively new class of antifungal drugs. Althoughthe most widely known echinocandin, caspofungin, is considered lesstoxic than other antifungal drugs, (e.g., Amphotericin B), this is nottrue of the entire class. Caspofungin is especially effective againstCandida species, however, other members of the echinocandin class haveactivity against other species, (e.g., Cryptococcus). Additionally,echinocandins are generally administered intravenously due to their poororal absorption. Cochleates of the present invention can be used notonly to facilitate oral absorption, but also to avoid potential sideeffects from this class of compounds.

Safety/Biocompatibility

Cochleates readily can be prepared from safe, simple, well-defined,naturally occurring substances, e.g., PS and calcium. Mixtures ofnaturally occurring (e.g., soy lipids), synthetic lipids, and/ormodified lipids can also be utilized. Phosphatidylserine is a naturalcomponent of all biological membranes, and is most concentrated in thebrain. The phospholipids used can be produced synthetically, or preparedfrom natural sources. Soy PS is inexpensive, available in largequantities and suitable for use in humans. Clinical studies indicatethat PS is safe and may play a role in the support of mental functionsin the aging brain. Unlike many cationic lipids, cochleates (which arecomposed of anionic lipids) are non-inflammatory and biodegradable. Thetolerance in vivo of mice to multiple administrations of cochleates byvarious routes, including intravenous, intraperitoneal, intranasal andoral, has been evaluated. Multiple administrations of high doses ofcochleate formulations to the same animal show no toxicity, and do notresult in either the development of an immune response to the cochleatematrix, or any side effects relating to the cochleate vehicle.

The cochleates and cochleate compositions of the present invention canbe administered to animals, including both human and non-human animals.It can be administered to animals, e.g., in animal feed or water. Forexample, antibiotic-cochleates of the present invention can beadministered to poultry and other farm animals, including the ruminantsand pigs, to control infection or to promote growth or milk production.Among a number of conditions which can be treated with these agents isenteritis, a disease which can cause severe economic losses to livestockproducers. Enteritis occurs in chickens, swine, cattle and sheep and isattributed mainly to anaerobic bacteria, particularly Clostridiumperfungens. Enterotoxemia in ruminants, an example of which is“overeating disease” in sheep, is a condition caused by C. perfungensinfection. The treatment of such conditions is therefore alsoencompassed within the methods of the present invention.

Methods of Treatment

In yet another aspect, the present invention provides for bothprophylactic and therapeutic methods of treating a subject at risk of(or susceptible to) a disorder or having a disorder which can be treatedwith one or more cargo moiety.

“Treatment”, or “treating” as used herein, is defined as the applicationor administration of a therapeutic agent (e.g., antibiotics encochleatedby cochleates of the invention) to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease or disorder, a symptom of disease ordisorder or a predisposition toward a disease or disorder, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease or disorder, the symptoms of the diseaseor disorder, or the predisposition toward disease or disorder.“Treated,” as used herein, refers to the disease or disorder beingcured, healed, alleviated, relieved, altered, remedied, amelioratedimproved or affected. For example, certain methods of treatment of theinstant invention provide for administration of anti-inflammatorycochleates, such that inflammation is lessened or alleviated. Othermethods of treatment of the instant invention include the administrationof antifungal cochleates, such that fungal infection is relieved orremedied.

The terms “cure,” “heal,” “alleviate,” “relieve,” “alter,” “remedy,”“ameliorate,” “improve” and “affect” are evaluated in terms of asuitable or appropriate control. A “suitable control” or “appropriatecontrol” is any control or standard familiar to one of ordinary skill inthe art useful for comparison purposes. In one embodiment, a “suitablecontrol” or “appropriate control” is a value, level, feature,characteristic, property, etc. determined prior to administration of acargo moiety cochleate, as described herein. For example, the number ofcolony forming units can be determined prior to administering anechinocandin cochleate of the invention to a host. In anotherembodiment, a “suitable control” or “appropriate control” is a value,level, feature, characteristic, property, etc. determined in a subject,e.g., a control or normal subject exhibiting, for example, normaltraits. In yet another embodiment, a “suitable control” or “appropriatecontrol” is a predefined value, level, feature, characteristic,property, etc.

The methods of the present invention include methods of administering acargo moiety to a host, wherein the cargo moiety is associated with acochleate or cochleate composition of the invention. The cochleates andcochleate compositions of the present invention may be administeredorally, nasally, topically, intravenously, transdermally, buccally,sublingually, rectally, vaginally or parenterally.

The present invention provides a method for treating a subject thatwould benefit from administration of a composition of the presentinvention. Any therapeutic indication that would benefit from a cargomoiety, e.g., a drug or nutrient, can be treated by the methods of theinvention. Accordingly, the present invention provides methods oftreating a subject at risk for or having a disease or disorder which canbe treated with, for example, a protein, a small peptide, a bioactivepolynucleotide, an antibiotic, an antiviral, an anesthetic,antipsychotic, an anti-infectious, an antifungal, an anticancer, aimmunosuppressant, an immunostimulant, a steroidal anti-inflammatory, anon-steroidal anti-inflammatory, an antioxidant, an antidepressant whichcan be synthetically or naturally derived, a substance which supports orenhances mental function or inhibits mental deterioration, ananticonvulsant, an HIV protease inhibitor, a non-nucleophilic reversetranscriptase inhibitor, a cytokine, a tranquilizer, a mucolytic agent,a dilator, a vasoconstrictor, a decongestant, a leukotriene inhibitor,an anti-cholinergic, an anti-histamine, a cholesterol lipid metabolismmodulating agent or a vasodilatory agent. The method includes the stepof administering to the subject a composition of the invention, suchthat the disease or disorder is treated. The disease or disorder can be,e.g., inflammation, pain, infection, fungal infection, bacterialinfection, viral infection, parasitic disorders, an immune disorder,genetic disorders, degenerative disorders, cancer, proliferativedisorders, obesity, depression, hair loss, impotence, hypertension,hypotension, dementia, senile dementia, or malnutrition, acute andchronic leukemia and lymphoma, sarcoma, adenoma, carcinomas, epithelialcancers, small cell lung cancer, non-small cell lung cancer, prostatecancer, breast cancer, pancreatic cancer, hepatocellular carcinoma,renal cell carcinoma, biliary cancer, colorectal cancer, ovarian cancer,uterine cancer, melanoma, cervical cancer, testicular cancer, esophagealcancer, gastric cancer, mesothelioma, glioma, glioblastoma, pituitaryadenomas, schizophrenia, obsessive compulsive disorder (OCD), bipolardisorder, Alzheimer's disease, Parkinson's disease, cell proliferativedisorders, blood coagulation disorders, Dysfibrinogenaemia andhemophilia (A and B), autoimmune disorders, e.g., systemic lupuserythematosis, multiple sclerosis, myasthenia gravis, autoimmunehemolytic anemia, autoimmune thrombocytopenia, Grave's disease,allogenic transplant rejection, ankylosing spondylitis, psoriasis,scleroderma, uveitis, eczema, dermatological disorders, hyperlipidemia,hyperglycemia, and hypercholesterolemia.

Cochleates of the instant invention can also be used to promote greaterhealth or quality of life, for example limit cholesterol uptake orregulate lipid metabolism, weight gain, hunger, aging, or growth.Cosmetic effects such as wrinkle reduction, hair growth, pigmentation,or dermatologic disorders may also be treated. Cochleates may also treathereditary disease such as cystic fibrosis or muscular dystrophy.

The cochleates of the instant invention can be used to treat a varietyof inflammations, including headache, arthritis, rheumatoid arthritis,osteoarthritis, atherosclerosis, acute gout, acute or chronic softtissue damage associated with, e.g., a sports injury, tennis elbow,bursitis, tendonitis, acute or chronic back pain, such as a herniateddisc, carpal tunnel syndrome, glomerulonephritis, carditis, ulcerativecolitis, asthma, sepsis, and plantar fasciitis. The cochleates of theinvention can also be used to relieve pain resulting from surgery orother medical procedure. The cochleates of the instant invention canfurther be used to treat a variety of fungal infections, includingcandida, e.g., yeast infection, tinea, e.g., Athlete's foot, pityriasis,thrush, cryptococcal meningitis, histoplasmosis, and blastomycosis.

The cochleates of the instant invention can also be used to treat avariety of bacterial infections, including but not limited to moderateto severe lower respiratory tract infections, skin infections, biliarytract infections, bone infections, antibiotic prophylaxis,pseudomembranous enterocolitis, central nervous system infections (e.g.,meningitis and ventriculitis), intra-abdominal infections (e.g.,peritonitis), pneumonia, septicemia, soft tissue infections, neutropenicsepsis, joint infections, infective endocartidis, and urinary tractinfections.

Exemplary bacteria that can be treated with the antibiotic preparationof the present invention include, but are not limited to, Staphylococcusaureus, Staphylococcus epidermidis, Streptococcus pyogenes,Streptococcus pneumuoniae, Streptococcus Group D, Clostridiumperfungens, Haemophilus influenzae, Escherichia coli, Pseudomonasaeruginosa, and Klebsiella pneumoniae.

The cochleate compositions of the invention are demonstrated herein toeffectively mediate the presence of bacteria such as Pseudomona andStaphylococcus. One species, S. aureus, one of the leading causes ofhospital acquired infections, causes a wide variety of suppurativediseases, including superficial and deep abscesses, empyema, meningitis,purulent arthritis, and septicemia and endocarditis. In addition, itcauses two toxinoses: food poisoning and exfolative skin disease.

Staphyloccoci found in infected tissues are mainly locatedextracellularly. However, virulent staphylococci can survive withinleukocytes after phagocytosis and may protect themselves against thebactericidal action of antibiotics by means of their intracellularlocation. Intraphagocytic survival of S. aureus has also been observedin patients with various disorders of phagocytic functions. Certaininfections caused by S. aureus have the tendency to become recurrent,which is attributable to the intraphagocytic survival of small numbersof the organism. Antibiotics that are able to penetrate leukocytes havebeen shown to have superior clinical efficacy in such recurrent andpersistent staphylococcal infections.

Because intracellular residence of infectious agents can complicatetreatment, a complete cure can require the eradication of allintracellular bacteria. Therefore, a therapeutic approach that increasesthe intracellular antibiotic concentration may enhance the bactericidalkilling and ensure complete elimination of infection.

Pseudomonas aeruginosa infections occur in individuals with altered hostdefenses, including burn patients, persons with malignant or metabolicdisease, or those who have had prior instrumentation or manipulation.Prolonged treatment with immunosuppressive or antimicrobial drugs andradiation therapy also predispose individuals to Pseudomonas infections.P. aeruginosa is a frequent cause of life-threatening infection, and isthe most common cause of nosocomial gram-negative pneumonia, with anassociated mortality rate of less than 60%. Among immunocompromisedpatients, P. aeruginosa is a frequent cause of nosocomial bacteremia. Incystic fibrosis, P. aeruginosa chronically colonizes the lung,eventually causing respiratory failure and death.

In a preferred embodiment, antibacterial cochleates of the presentinvention have the ability to reduce the number of bacterial colonies byat least 10%. More preferably, antibacterial cochleates can reduce thenumber of bacterial colonies by at least 25% and even more preferably by50%, 75%, 85%, 95%, . . . 100%. All individual values and ranges fallingbetween these ranges and values are within the scope of the presentinvention.

The present invention also provides a means for treating a variety offungal infections, including, but not limited to, asthma, chronicrhinosinusitis, allergic fungal sinusitis, sinus mycetoma, non-invasivefungus induced mucositis, non-invasive fungus induced intestinalmucositis, chronic otitis media, chronic colitis, inflammatory boweldiseases, ulcerative colitis, Crohn's disease, candidemia,intraabdominal abscesses, peritonitis, pleural space infections,esophageal candidiasis and invasive aspergillosis. Exemplary fungi thatcan be treated using antifungal cochleates of the invention include,without limitation, Absidia, Aspergillus flavus, Aspergillus fumigatus,Aspergillus glaucus, Aspergillus nidulans, Aspergillus terreus,Aspergillus versicolor, Alternaria, Basidiobolus, Bipolaris, Candidaalbicans, Candida glabrata, Candida guilliermondii, Candida krusei,Candida lypolytica, Candida parapsilosis, Candida tropicalis,Cladosporium, Conidiobolus, Cunninahamella, Curvularia, Dreschlera,Exserohilum, Fusarium, Malbranchia, Paecilonvces, Penicillium,Pseudallescheria, Rhizopus, Schizophylum, Sporothrix, Acremonium,Arachniotus citrinus, Aurobasidioum, Beauveria, Chaetomium,Chryosporium, Epicoccum, Exophilia jeanselmei, Geotrichum, Oidiodendron,Phoma, Pithomyces, Rhinocladiella, Rhodoturula, Sagrahamala,Scolebasidium, Scopulariopsis, Ustilago, Trichoderma, and Zygomycete.

Candida albicans is part of the normal microbial flora that colonizesmucocutaneous surfaces of the oral cavity gastrointestinal tract, andvagina of many mammals and birds. Because both antibody- andcell-mediated immune responses to Candida antigens are evoked in healthyindividuals, C. albicans colonies are generally infectious for the host.C. albicans, however, does not normally cause disease in immunocompetentcolonized hosts. It is the setting of congenital, induced, ordisease-related immune dysfunction that C. albicans causes cutaneous,mucocutaneous, and life-threatening systemic disease.

C. albicans is able to not only compete with other microbes but alsoadhere to and survive on mucosal surfaces of a host withCandida-specific antibody and cell-mediated immunity. Numerous putativeC. albicans virulence factors exist that may enable this opportunisticfungus to survive and thrive in the adverse conditions of host tissues.Among these putative virulence factors, the cell wall of C. albicans isone of the most important. The cell wall provides rigidity as well asprotection against osmotic lysis, and it promotes infection bysupporting the interaction of C. albicans adhesins and host-cellreceptors. Also, the C. albicans cell wall contains mannoproteins whichhave immunosuppressive properties that can enhance the persistence ofthe fungus in lesions. Echinocandins, unlike other antifungals, functionby interfering with the synthesis of the fungal cell wall.

In a preferred embodiment, echinocandin cochleates of the presentinvention have the ability to reduce fungal colony forming units (CFU's)by at least 10%. More preferably, echinocandin cochleates can reduceCFU's by at least 25% and even more preferably by 50%, 75%, 85%, 95%, .. . 100%. All individual values and ranges falling between these rangesand values are within the scope of the present invention. Reduction incolony forming units may be in vivo or in vitro. The host of the fungalinfection can be a human or non-human animal.

Macrophages are important in the uptake of bacteria, fungi andparasites, and also play an important role in the inflammatory response.In addition to performing phagocytosis, macrophages have the potentialof being activated, a process that results in increased cell size,increased levels of lysosomal enzymes, more active metabolism, andgreater ability to phagocytose and kill ingested microbes. Afteractivation, macrophages secrete a wide variety of biologically activeproducts that, if unchecked, result in tissue injury and chronicinflammation. One of the secreted products, nitric oxide (NO) has comeinto the forefront as a mediator of inflammation.

Nitric oxide (NO) produced by inducible NOS plays an important role ininflammation, killing of bacterial pathogens, and tissue repair. NOformation increases during inflammation (i.e., in rheumatoid arthritis,ulcerative colitis, and Crohns disease), and several classicinflammatory symptoms, (i.e., erythema and vascular weakness) arereversed by NOS inhibitors. Nitric oxide has also been recognized asplaying a versatile role in the immune system. It is involved in thepathogenesis and control of infectious diseases, tumors, autoimmuneprocesses and chronic degenerative diseases.

Aspirin and acetaminophen are used as anti-inflammatory drugs to relievepain and fever. The mechanism of action and side effects of these drugsare explained in part by the generation of NO from iNOS. Inhibition ofiNOS expression and NO production, therefore, could be a way totherapeutically decrease the inflammatory actions of these drugs.

The above methods can be employed in the absence of other treatment, orin combination with other treatments. Such treatments can be startedprior to, concurrent with, or after the administration of thecompositions of the instant invention. Accordingly, the methods of theinvention can further include the step of administering a secondtreatment, such as for example, a second treatment for the disease ordisorder or to ameliorate side effects of other treatments. Such secondtreatment can include, e.g., radiation, chemotherapy, transfusion,operations (e.g., excision to remove tumors), and gene therapy.Additionally or alternatively, further treatment can includeadministration of drugs to further treat the disease or to treat a sideeffect of the disease or other treatments (e.g., anti-nausea drugs).

With regard to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket.

More specifically, the term refers the study of how a patient's genesdetermine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment according to that individual'sdrug response genotype. Pharmacogenomics allows a clinician or physicianto target prophylactic or therapeutic treatments to patients who willmost benefit from the treatment and to avoid treatment of patients whowill experience toxic drug-related side effects.

The language “therapeutically effective amount” is that amount necessaryor sufficient to produce the desired physiologic response. The effectiveamount may vary depending on such factors as the size and weight of thesubject, or the particular compound. The effective amount may bedetermined through consideration of the toxicity and therapeuticefficacy of the compounds by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., for determining the LD₅₀ (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itmay be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit largetherapeutic indices are preferred. While compounds that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue inorder to minimize potential damage to unaffected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compositionused in the method of the invention, the therapeutically effective dosecan be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the EC50 (i.e., the concentration ofthe test composition that achieves a half-maximal response) asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or disorder which can be treated with at least onecargo moiety, e.g., a protein, a small peptide, an antiviral, ananesthetic, an anti-infectious, an antifungal, an anticancer, animmunosuppressant, a steroidal anti-inflammatory, a non-steroidalanti-inflammatory, a tranquilizer, a mucolytic agent, a dilator, avasoconstrictor, a decongestant, a leukotriene inhibitor, ananti-cholinergic, an anti-histamine or a vasodilatory agent. Subjects atrisk for a disease or condition which can be treated with the agentsmentioned herein can be identified by, for example, any or a combinationof diagnostic or prognostic assays known to those skilled in the art.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the disease or disorder,such that the disease or disorder is prevented or, alternatively,delayed in its progression. Amphotericin B cochleates, for example, havebeen administered prophylactically in mice, and were at least asefficacious, if not more efficacious, than Amphotericin B deoxycholate.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of administering acochleate composition for therapeutic purposes. In one embodiment, thepresent invention provides a method for treating a subject that wouldbenefit from administration of a composition of the present invention.Any therapeutic indication that would benefit from a cochleatecomposition of the invention can be treated by the methods of theinvention. The present invention provides methods of treating a subjectat risk for or having a disease or disorder that can be treated with oneor more cargo moiety. The method includes the step of administering tothe subject a composition of the invention, such that the disease ordisorder is prevented, ameliorated, terminated or delayed in itsprogression. The disease or disorder can be any of the diseases ordisorders discussed herein.

The compositions of the invention can be administered to a subject aloneor in combination with a second therapy as described above. Thecompositions of the invention can be administered to a subject prior to,at the same time, or after a second therapy is administered.

Therapeutic agents can be tested in an appropriate animal model. Forexample, cochleate compositions of the present invention can be used inan animal model to determine the efficacy, toxicity, or side effects oftreatment with said agent. Alternatively, a therapeutic agent can beused in an animal model to determine the mechanism of action of such anagent. For example, an agent can be used in an animal model to determinethe efficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent can be used in an animal model to determine themechanism of action of such an agent.

Pharmaceutical Compositions

The invention pertains to uses of the cochleate compositions of theinvention for prophylactic and therapeutic treatments as describedinfra. Accordingly, the compounds of the present invention can beincorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the compositions ofthe invention and a pharmaceutically acceptable carrier. As used hereinthe language “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, sweetening, flavoring and perfuming agents, preservatives andantioxidants may also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants, which may also bepresent in formulations of therapeutic compounds of the invention,include water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Furthermore, the present invention can further include one or moreadditional agents, including water, antimicrobial agents, plasticizingagents, flavoring agents, surfactants, stabilizing agents, emulsifyingagents, thickening agents, binding agents, coloring agents, sweeteners,fragrances, and the like.

Suitable antimicrobial agents include triclosan, cetyl pyridiumchloride, domiphen bromide, quaternary ammonium salts, zinc compounds,sanguinarine, fluorides, alexidine, octonidine, EDTA, and essential oilssuch as thymol, methyl salicylate, menthol and eucalyptol.

Suitable plasticizing agents include, for example, polyols such assugars, sugar alcohols, or polyethylene glycols (PEGs), urea; glycol,propylene glycol, triethyl citrate, dibutyl or dimethyl phthalate,monoacetin, diacetin or triacetin.

Suitable surfactants include pluronic acid, sodium lauryl sulfate, monoand diglycerides of fatty acids and polyoxyethylene sorbitol esters,such as, Atmos 300 and Polysorbate 80. Suitabie stabilizing agentsinclude xanthan gum, locust bean gum, guar gum, and carrageenan.Suitable emulsifying agents include triethanolamine stearate, quaternaryammonium compounds, acacia, gelatin, lecithin, bentonite, veegum, andthe like. Suitable thickening agents include methylcellulose, carboxylmethylcellulose, and the like. Suitable binding agents include starch.

Suitable sweeteners that can be included are those well known in theart, including both natural and artificial sweeteners. Suitablesweeteners include water-soluble sweetening agents such asmonosaccharides, disaccharides and polysaccharides; water-solubleartificial sweeteners such as soluble saccharin salts, cyclamate salts,or the free acid form of saccharin, and the like; dipeptide basedsweeteners, such as L-aspartic acid derived sweeteners; water-solublesweeteners derived from naturally occurring water-soluble sweeteners,such as a chlorinated derivative of ordinary sugar (sucrose), known,under the product description of sucralose; and protein based sweetenerssuch as thaumatoccous danielli (Thaumatin I and II).

In general, an effective amount of auxiliary sweetener is utilized toprovide the level of sweetness desired for a particular composition, andthis amount will vary with the sweetener selected. This amount willnormally be 0.01% to about 10% by weight of the composition when usingan easily extractable sweetener.

The flavorings that can be used include those known to the skilledartisan, such as natural and artificial flavors. These flavorings may bechosen from synthetic flavor oils and flavoring aromatics, and/or oils,oleo resins and extracts derived from plants, leaves, flowers, fruitsand so forth, and combinations thereof. Representative flavor oilsinclude: spearmint oil, cinnamon oil, peppermint oil, clove oil, bayoil, thyme oil, cedar leaf oil, oil of nutmeg, oil of sage, and oil ofbitter almonds. Also useful are artificial, natural or synthetic fruitflavors such as vanilla, chocolate, coffee, cocoa and citrus oil, andfruit essences. These flavorings can be used individually or inadmixture. Flavorings such as aldehydes and esters including cinnamylacetate, cinnamaldehyde, citral, diethylacetal, dihydrocarvyl acetate,eugenyl formate, p-methylanisole, and so forth may also be used.Generally, any flavoring or food additive, such as those described inChemicals Used in Food Processing, publication 1274 by the NationalAcademy of Sciences, pages 63-258, may be used.

The amount of flavoring employed is normally a matter of preferencesubject to such factors as flavor type, individual flavor, and strengthdesired. Thus, the amount may be varied in order to obtain the resultdesired in the final product. Such variations are within thecapabilities of those skilled in the art without the need for undueexperimentation.

The compositions of this invention can also contain coloring agents orcolorants. The coloring agents are used in amounts' effective to producethe desired color. The coloring agents useful in the present inventioninclude pigments such as titanium dioxide, which may be incorporated inamounts of up to about 5 wt %, and preferably less than about 1 wt %.Colorants can also include natural food colors and dyes suitable forfood, drug and cosmetic applications. These colorants are known as FD&Cdyes and lakes. A full recitation of all FD&C and D&C dyes and theircorresponding chemical structures may be found in the Kirk-OthmerEncyclopedia of Chemical Technology, Volume 5, Pages 857-884, which textis accordingly incorporated herein by reference.

Formulations of the present invention include those suitable for oral,nasal, topical, transdermal, buccal, sublingual, rectal, vaginal orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which maybe combined with a carrier material to produce a single dosage form willgenerally be that amount of the composition which produces a therapeuticeffect. Generally, out of 100%, this amount will range from about 1% toabout 99% of active ingredient preferably from about 5% to about 70%,most preferably from about 10% to about 30%.

Methods of preparing these formulations or compositions include the stepof bringing into association a composition of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a composition of the present invention withliquid carriers, or finely divided solid carriers, or both, and then ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, gelcaps, crystallinesubstances, lozenges (using a flavored basis, usually sucrose and acaciaor tragacanth), powders, granules, gel, partial liquid, spray, nebulae,mist, atomized vapor, aerosol, tincture, or as a solution or asuspension in an aqueous or non-aqueous liquid, or as an oil-in-water orwater-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles(using an inert base, such as gelatin and glycerin, or sucrose andacacia) or as mouth washes and the like, each containing a predeterminedamount of a composition of the present invention as an activeingredient. A composition of the present invention may also beadministered as a bolus, electuary, or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules, and the like),the active ingredient is mixed with one or more pharmaceuticallyacceptable carriers, such as sodium citrate or dicalcium phosphate, orany of the following: fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, or silicic acid; binders, such as, forexample, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose or acacia; humectants, such as glycerol;disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;solution retarding agents, such as paraffin; absorption accelerators,such as quaternary ammonium compounds; wetting agents, such as, forexample, cetyl alcohol and glycerol monostearate; absorbents, such askaolin and bentonite clay; lubricants, such a talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl-sulfate,and mixtures thereof; and coloring agents.

In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing-agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compositionmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes or microspheres.

They may be sterilized by, for example, filtration through abacteria-retaining filter, or by incorporating sterilizing agents in theform of sterile solid compositions which may be dissolved in sterilewater, or some other sterile injectable medium immediately before use.

These compositions may also optionally contain opacifying agents and maybe of a composition that they release the active ingredient(s) only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich may be used include polymeric substances and waxes. The activeingredient may also be in micro-encapsulated form, if appropriate, withone or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert dilutents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof. Besides inert dilutents, theoral compositions may also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, coloring,perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented in liquid or aerosolform, or as a suppository, which may be prepared by mixing one or morecompounds of the invention with one or more suitable nonirritatingexcipients or carriers comprising, for example, cocoa butter,polyethylene glycol, a suppository wax or a salicylate, and which issolid at room temperature, but liquid at body temperature and,therefore, will melt in the rectum or vaginal cavity and release theactive compound. Liquid or aerosol forms include, but are not limitedto, gels, pastes, ointments, salves, creams, solutions, suspensions,partial liquids, sprays, nebulaes, mists, atomized vapors, andtinctures. Formulations of the present invention which are suitable forvaginal administration also include pessaries, tampons, creams, gels,pastes, foams or spray formulations containing such carriers as areknown in the art to be appropriate.

Formulations of the pharmaceutical compositions of the invention fornasal administration can be in solid, liquid, or aerosol form (e.g.,powder, crystalline substance, gel, paste, ointment, salve, cream,solution, suspension, partial liquid, spray, nebulae, irrigant, wash,mist, atomized vapor or tincture).

Dosage forms for the topical or transdermal administration of acomposition of this invention include powders, sprays, ointments,pastes, creams, lotions, gels, solutions, patches and inhalants. Thecomposition may be mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to ancomposition of this invention, excipients, such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays may contain, in addition to a composition of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays may additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a composition of the present invention to the body. Suchdosage forms may be made by dissolving or dispersing the composition inthe proper medium. Absorption enhancers may also be used to increase theflux of the composition across the skin. The rate of such flux may becontrolled by either providing a rate controlling membrane or dispersingthe composition in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the likeare also within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise a cochleate or cochleate composition of theinvention in combination with one or more pharmaceutically acceptablesterile isotonic aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, or sterile powders which may be reconstitutedinto sterile injectable solutions or dispersions just prior to use,which may contain antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity may be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, and sodium chloride, inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating acomposition of the invention in the desired amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the composition into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe cochleate compositions of the invention plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease may be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, thecomposition can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the composition inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal-administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compositions of the invention also can be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the compositions of the invention are prepared withcarriers that will protect the composition against rapid eliminationfrom the body, such as a controlled release formulation, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art. The materials can also be obtainedcommercially from Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of acomposition calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the composition and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such a composition for the treatmentof individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The pharmaceutical compositions can be included in a container alongwith one or more additional compounds or compositions and instructionsfor use. For example, the invention also provides for packagedpharmaceutical products containing two agents, each of which exerts atherapeutic effect when administered to a subject in need thereof. Apharmaceutical composition may also comprise a third agent, or even moreagents yet, wherein the third (and fourth, etc.) agent can be anotheragent against the disorder, such as a cancer treatment (e.g., ananticancer drug and/or chemotherapy) or an HIV cocktail. In some cases,the individual agents may be packaged in separate containers for sale ordelivery to the consumer. The agents of the invention may be supplied ina solution with an appropriate solvent or in a solvent-free form (e.g.,lyophilized). Additional components may include acids, bases, bufferingagents, inorganic salts, solvents, antioxidants, preservatives, or metalchelators. The additional kit components are present as purecompositions, or as aqueous or organic solutions that incorporate one ormore additional kit components. Any or all of the kit componentsoptionally further comprise buffers.

The present invention also includes packaged pharmaceutical productscontaining a first agent in combination with (e.g., intermixed with) asecond agent. The invention also includes a pharmaceutical productcomprising a first agent packaged with instructions for using the firstagent in the presence of a second agent or instructions for use of thefirst agent in a method of the invention. The invention also includes apharmaceutical product comprising a second or additional agents packagedwith instructions for using the second or additional agents in thepresence of a first agent or instructions for use of the second oradditional agents in a method of the invention. Alternatively, thepackaged pharmaceutical product may contain at least one of the agentsand the product may be promoted for use with a second agent.

In yet another aspect, the invention provides an article of manufactureof cochleates and/or cochleate compositions of the invention (FIG. 29).The article of manufacture includes packaging material and a lipidcontained within the packaging material. The packaging material includesa label or package insert indicating the use of the lipid for formingcochleates or cochleate compositions of the invention. The article ofmanufacture can further include instructions or guidelines for theformation of cochleates or cochleate compositions of the invention,e.g., mixing a cargo moiety with a solvent and dripping it into asolution of the lipids. Optionally, the article of manufacture caninclude a solvent, a cargo moiety, a multivalent cation (e.g., calciumand/or magnesium), a control cargo moiety, and/or a chelating agent(e.g., EDTA).

The article of manufacture may further include other ingredients orapparatus that can be employed to manufacture the compositions of thepresent invention. One non-limiting example of an article of manufacturewould include 5 g of powdered Soy PS, a solution of a model hydrophobiccompound in DMSO as a positive control, a solution of calcium chlorideto induce cochleate formation, and a solution of EDTA to visualize theopening of the cochleates into liposomes.

The instructions and/or guidelines may generally include one or more ofthe following statements:

1. Prepare a liposomal suspension by vigorously mixing lipid in water orbuffer.

2. Monitor lipid concentrations: low concentration would require a largevolume of buffer in order to formulate an adequate amount of end productand high concentration may produce large cochleate aggregates upon theaddition of calcium.

3. Experimentally determine whether to use water or buffered solution:the presence of salts and the pH of the suspension may affect theformation of the cargo moiety-liposome intermediate depending on theproperties of the cargo moiety.

4. Optionally filter or perform other standard procedures to prepareliposomes of defined size and/or to sterilize the suspension.

5. Prepare a cargo moiety solution with an appropriate solvent: manysolvents may potentially be used in this process, e.g., DMSO.

6. Add the cargo moiety-solvent solution, preferably dropwise, to theliposome suspension with vigorous mixing.

7. Experimentally determine the optimal rate of addition and speed ofmixing: a suspension of cargo Moiety-liposomes essentially free ofunencochleated cargo moiety when viewed by light microscopy should beproduced.

8. Calculate the amount of calcium to be added by assuming one mole ofcalcium for every two moles of lipid, and adding extra calcium to bringthe buffer to between 2 and 6 mM.

9. Induce cochleate formation through addition of a calcium salt. Thesalt may be added as a solution, e.g., 0.1 M calcium chloride, or may beslowly added as a solid calcium salt, e.g., calcium chloride, withvigorous mixing.

10. If the presence of solvent in the buffer is unwanted, optionallyharvest the cargo moiety-cochleates, e.g., by centrifugation orfiltration, and resuspending them in an appropriate medium. Theassociation of calcium ions with PS is easily reversible, therefore, inorder to remain intact and in their crystalline state, cochleateformulations can be resuspended in a medium containing at least 1 to 2mM calcium ions.

11. Optionally evaluate the quality of the cochleate formulation. Thepresence of sufficient calcium ions initiates and maintains thecochleate structure. One method of evaluating the quality of a cochleateformulation is visualization of the liposomes that are produced uponremoval of the calcium ions from a cochleate crystal. This may beaccomplished using light microscopy. An aliquot of a cargomoiety-cochleate suspension may be visualized by phase contrastmicroscopy at 1000× magnification. A small amount of a concentratedsolution of a chelating agent, e.g., EDTA, may be added to the edge ofthe cover slip, thus reaching the sample through capillary action. Ahigh-quality cochleate product will open into intact liposomes uponcontact with the calcium-chelating agent. When using EDTA as thechelating agent, the pH of the EDTA solution should be about pH 9.5.Cochleates will not convert to liposomes at a pH below 6.5. If EDTAsolutions at pH 7.4 are used, the release of hydrogen ions upon thebinding of calcium to the acetate groups of the chelating agent lowersthe pH of the solution and inhibits cochleate conversion to liposomes.

Choice of solvent and other materials, optimal rate of dropwiseaddition, speed of mixing, the amount of calcium, etc., can readily bedetermined by the skilled practitioner employing the teachings providedherein.

In addition, a skilled practitioner can introduce, modify and/oreliminate elements and/or steps to the above without departing from thescope of the invention. For example, a liposome suspension might beprovided already prepared, a combination of solvents might be used,excess calcium might be used to obviate the calculation of calcium,alternative or additional cations might be employed, etc.

Practice of the invention will be still more fully understood from thefollowing examples, which are presented herein for illustration only andshould not be construed as limiting in any way.

EXEMPLIFICATION

Materials and Methods

Materials

The following materials were used, unless otherwise indicated: powderedAmphotericin B (AmB) U.S. Pharmacopela grade was obtained from USP(Rockville, Md.) and Alpharma (Copenhagen, Denmark), and stored at 4°C.; powdered Soy PS was obtained from American Lethicin Corporation(Oxford, Conn.) and Degussa (Champaign, Ill.) and stored at roomtemperature; Vitamin E (V-E) was obtained from Roche (Parsippany, N.J.);sterile water was obtained from Baxter (Canada); Dioleoylphosphatidylserine (DOPS) was obtained from Avanti Polar Lipid (Alabaster, Ala.),methyl sulfoxide (DMSO)HPLC grade was obtained from Aldrich (Milwaukee,Wis.), and, micellar AmB/deoxycholate suspension sold under thetrademark FUNGIZONE was obtained from Sigma (St. Louis, Mo.).

General Method for Forming Cochleates with a Cargo Moiety

Lipid powder (soy PS or synthetic PS) is dispersed in water (pure wateror saline) by vortexing, resulting mixture of unilamellar andmultilamellar liposomes. The liposomal suspension is filtered to obtaina suspension having a majority of unilamellar liposomes. To thisliposome suspension, a water miscible organic solvent (e.g., DMSO)including a cargo moiety (and optional antioxidant) is introduced. Theliposomal suspension is precipitated with cation. The solvent may beremoved from the liposomal suspension by tangential flow and/orfiltration and/or dialysis, or from the cochleates by washing,filtration, centrifugation, tangential flow, and/or dialysis.

Cell Lines and Culture Conditions

Mouse macrophage J774A.1 cell line and ovarian cancer cell line SKOV3cell line were obtained from ATCC and PPD, respectively. The cells weregrown in monolayers in humidified air with 5% CO₂ at 37° C. in 60 mm²Petri dishes (Corning) containing 5 mL of DMEM supplemented with 10%FBS. For experiments, cells were harvested by scraping (J774A.1) ortrypsinization (SKOV3), and were seeded into 24 or 96-well plates at adensity of 5×10⁵ cells.

Staphylococcal aureus (ATCC 29213) and Pseudomonas aeruginosa (ATCC700289) were maintained weekly on Nutrient agar plates and slants. Freshcultures were grown up to 24 hours prior to experiment.

Imaging

Phase contrast light microscopy and confocal microscopy (Olympus) wasused to image liposomal suspensions, cochleates and cells, with andwithout the aid of fluorescence, which can be used, e.g., to studycellular uptake and intracellular distribution of fluorescently labeledcochleates and cargo moieties. Confocal microscopy is particularlyadvantageous as it is a 3-dimensional digital imaging device that can beused to effectively view slices of cell culture. This allowsverification of the presence of cochleate and other agents within acell.

Particle Size Analysis

Two different devices were used to examine particle size. The N4 plus(Coulter) measures particles in the range 10 nm to 3000 nm. The LS230(Beckman/Coulter) measures particles in the range 40 nm to 1 mm. Usingthe two devices provides the flexibility and capability to evaluateformulations ranging from nanocochleates to large aggregates ofcochleates.

Fraunhofer was used as the optical model for all the experiments. Theoptical models used to calculate absolute particle size were forspherical particles. Since cochleates are not spherical, the numbersgiven are relative, not absolute values, but nonetheless allow batch tobatch sample comparisons. The results obtained by the two differentdevices give a qualitative comparison of the size differences betweenthe formulations. However, light and electron microscopy have confirmedthat the “nanocochleates” obtained are submicron in size.

Example 1 Amphotericin B Cochleate (CAMB) Prepared with DMSO andLipid:AmB Ratios of 10:1, 2:1 and 1:1 w/w

Amphotericin B cochleates (CAMB) were prepared with Soy PS and DMSO withVitamin E, and a Lipid to AmB ratio of 10:1 as follows.

Preparation of Liposomes

20 ml of water was added to 200 mg of Soy-PS in a 50 ml plastic tube,vortexed for about 15 minutes to form a liposomal suspension, andfiltered using a 0.45 μm filter. The suspension was sonicated for about4 minutes and filtered again with a 0.22 μm filter.

Addition of Cargo Moiety and Antioxidant in Solvent

1.90 ml of DMSO solvent was added to 20 mg of Amphotericin B in a 15 mlplastic tube. To the AmB/DMSO mixture, 0.1 ml of Vitamin E (20 mg/ml inDMSO), vortexed for about 10 minutes. This solution was then added tothe liposomal suspension by drop wise addition using a 1 ml pipettewhile vortexing. The final mixture was vortexed for about 2 minutes.

Precipitation of Cochleates

2 ml of calcium (0.1 N) was added to the liposomal suspension at a rateof 10 μl/10 s while vortexing to form cochleates.

Solvent Removal/Washing

The mixture was vortexed for about 1-2 minutes, centrifuged for about 1hours at 9000 rpm, and the supernatant was removed and replaced withfresh supernatant (water with 2 mM calcium). This washing step wasrepeated once.

Employing the above method, cochleates having a lipid to drug ratio 2:1and 1:1 also were prepared by varying the ingredients to conform tofollowing formulations.

TABLE 1 Cochleate Formulations CAMB CAMB 1:1 2:1 Soy PS (mg) 100 200 AmB(mg) 100 100 Vitamin E (mg) 1 2 DMSO (ml) 2 2 Water (ml) 10 20 Calcium(ml) 1 2 Washings with sterile water w/calcium (1 mM) 2 2 Final Volume(ml) 10 20Summary of Results

For each cochleate formulation, a yellowish suspension with some of thecochleates floating on the top and/or residing on the bottom of thesuspension were observed macroscopically.

FIG. 4 is a series of images of the formulation having a 1:1 ratio atdifferent stages in the formulation: liposomes, liposomes with AmB,cochleates, and cochleates after addition of EDTA. FIGS. 5, 6 and 7 areeach a series of images, before and after addition of EDTA, of thecochleate formulations having a 10:1, 2:1, and 1:1 ratio, respectively.

FIG. 8 is a graph of the size distribution of the liposomes aftervortexing and prior to filtration, after filtration with 0.45 μm filter,and after introducing DMSO/Amphotericin. FIG. 9 is a graph of the sizedistribution of cochleate formulations having lipid to AmB ratios of10:1, 2:1 and 1:1.

Example 2 Amphotericin B Cochleates Prepared with NMP

Cochleates were prepared as described in Example 1, exceptN-methylpyrrolidone (NMP) solvent was used instead of DMSO, and theformulation was adjusted as indicated in the following table.

TABLE 2 NMP 10:1 Formulation CAMB/NMP 10:1 Soy PS (mg) 200 AmB (mg) 20Vitamin E (mg) 2 DMSO (ml) 2 Water (ml) 20 Calcium (ml) 2 Washings withsterile water w/calcium (1 mM) 2 Final Volume 20 ml

Cochleates in the final formulations were observed as a yellowishsuspension. Mice infected with a lethal dose of Aspergillus fumigatuswere dosed with 2 mg/kg of the AmB-cochleate formulation for 14 days.The cochleates were efficacious against the A. fumigatus.

Example 3 Amphotericin B Cochleates Prepared with DMSO and Lipid:AmBRatios of 5:1, 4:1, 3:1 and 2:1 w/w

Amphotericin B cochleates (CAMB) were prepared with soy PS and DMSO withtocopherol (Vitamin E) with the following protocol:

-   1. Weighing and placing 300 mg of soy PS into a 50 ml pp sterile    tube with 10 ml sterile water.-   2. Vortexing the suspension for 2 minutes.-   3. Sonicating the suspension for 3 minutes.-   4. Filtering the suspension with a 0.22 μm filter and pooling    liposomes into a 50 ml tube.-   5. Weighing and placing 10 mg (5:1), 12.5 mg (4:1) 16.6 mg (3:1) and    25 mg (2:1), of AmB (individually) into four 15 ml pp sterile tubes    with 0.5 ml DMSO and vortexing.-   6. Adding 6.0 μl (5:1), 6.2 μl (4:1), 6.6 μl (3:1), and 7.5 μl (2:1)    of tocopherol at 10 mg/ml in DMSO to the 15 ml tubes with AmB. (The    concentration of the AmB was 20 mg/ml 5:1), 25 mg/ml (4:1), 33.2    mg/ml (3:1), and 50 mg/ml (2:1), at this time)-   7. Vortexing the solution for a few minutes until the AmB completely    dissolved.-   8. Adding 5 ml of liposomes to each AmB/Vitamin E/DMSO solution, and    vortexing the sample for a few minutes.-   9. Adding 0.5 ml of 0.1M calcium solution into the suspension with    vortexing, using an eppendorf repeater pipette with a 500 μl tip and    adding 10 μl aliquots to the tube per every 10 sec.-   10. Centrifuging the suspension for 30 minutes at 9000 rpm at 4° C.-   11. Removing the supernatant from the tube and re-suspending the    pellet with the same volume of wash buffer (sterile water with 2 mM    calcium).-   12. Repeating steps 10 and 11 two more times. Adjusting the final    volume of the suspension to 6 ml with wash buffer.-   13. Examining the final preparation under a microscope and    confirming the pH of 5.5.-   14. Streaking the sample on a chocolate plate to check the    sterilization of the final preparation and incubating plates at 37°    C., 4° C., and room temperature for 24 hrs, 48 hrs, and 72 hrs.-   15. Storing the sample, treated with nitrogen and covered with    parafilm, at 4° C.

The following table summarizes the above formulations.

TABLE 3 CAMB Preparations Name of PS:AmB AmB 0.1M Final AmB Sample (w/w)AmB Soy PS V-E liposome Calcium (mg/ml) CAMB 5:1 5:1 10 mg 50 mg 60 μg5.5 ml 0.5 ml 1.66 mg/ml CAMB 4:1 4:1 12.5 mg  50 mg 62.5 μg  5.5 ml 0.5ml 2.08 mg/ml CAMB 3:1 3:1 16.6 mg  50 mg 66.6 μg  5.5 ml 0.5 ml 2.76mg/ml CAMB 2:1 2:1 25 mg 50 mg 75 μg 5.5 ml 0.5 ml 4.16 mg/ml

About 0.5 ml DMSO was used in each preparation. HPLC and LC/MS were usedto measure the AmB and DMSO concentrations.

Summary of Results

-   1. Macroscopic observations: yellowish suspension.-   2. Microscopic observations: cochleates with different size of    aggregates.-   3. Addition of EDTA: liposomes formed after addition of EDTA    (chelator).-   4. Recovery: HPLC analysis indicated that approximately 100% of the    AMB was successfully encochleated.-   5. For the mouse study described in Example 4, the following amounts    of each formulation were set aside:    -   5:1=>0.4 mg/ml×1.2 ml, 14 bottles    -   4:1=>0.4 mg/ml×1.2 ml, 14 bottles    -   3:1=>0.4 mg/ml×1.2 ml, 14 bottles    -   2:1=>0.4 mg/ml×1.2 ml 14 bottles

For the in vitro study described in Example 5, the following amounts ofeach formulation were set aside:

-   -   5:1=>0.34 mg/ml×100 ul (Conc: PS=1.7 mg/ml)    -   4:1=>0.42 mg/ml×100 ul (Conc: PS=1.7 mg/ml)    -   3:1=>0.56 mg/ml×100 ul (Conc: PS=1.7 mg/ml)    -   2:1=>0.85 mg/ml×100 ul (Conc: PS=1.7 mg/ml)

-   6. Sterility: No bacteria observed in the formulations after 72 hrs.

Example 4 Efficacy Studies in Mice

The formulations of Example 3 were administered to mice to study theefficacy of the formulations to protect mice from a lethal dose ofCandida albicans, and to clear the organs of C. albicans in thesurviving mice.

Six groups of 10 mice were studied. The mice were administered 5×10⁵cells C. albicans intravenously through the tail vein. Starting 24 hourspost-infection, the following compositions were administered to eachgroup orally once daily at 2 mg AmB/kg body weight for 14 days, exceptfor the control group which remained untreated.

-   -   a. Control (untreated)    -   b. AmB/deoxycholate (FUNGIZONE)    -   c. CAMB 2:1 AmB    -   d. CAMB 3:1 AmB    -   e. CAMB 4:1 AmB    -   f. CAMB 5:1 AmB

Appearance and behavior was monitored each day of the study. Tissueburden of C. Albicans was determined in kidney, liver and lungs for eachanimal, and colony counts were taken. Organs were obtained and weighed,homogenized, dilutions in buffer made, and aliquots plated onto plates;colony counts of fungus were taken several days later. FIG. 10 is agraph of the survival data for C. albicans-infected mice untreated(control), or dosed daily for 14 days with AmB/deoxycholate, orAmB-cochleates with a lipid to drug ratio of 2:1, 3:1, 4:1, or 5:1.

FIG. 11 is a chart of the average number of C. albicans cells/gram oftissue in the liver, kidney, and lungs of C. albicans-infected miceuntreated and dosed daily for 14 days with AmB/deoxycholate, orAmB-cochleates with a lipid to drug ratio of 2:1, 3:1, 4:1, or 5:1.

One hundred percent of the control (untreated) group did not survive thestudy and showed high tissue burdens. All four AmB-cochleate (CAMB)formulations were effective in preventing mortality and reducing fungalcell burdens in target organs (kidneys, lungs and liver). The CAMB 5:1and CAMB 3:1 formulation appeared somewhat better than the othersclearing the liver and lungs completely (the principal target organ forC. albicans (kidney) was not completely cleared). The CAMB 5:1formulation appeared to be the most effective versus the others inreducing fungal cell burdens in the kidneys. In general, the CAMBformulations were more effective than the AmB/deoxycholate formulation.

Example 5 Efficacy Studies in Cells

The relative efficacy of the compositions of Example 3 were studied inJ774A.1 macrophages to compare the relative efficacy of the cochleatecompositions (5:1, 4:1, 3:1 and 2:1) against Candida albicans.

Macrophages were seeded into a 96-well plate and incubated overnight asdescribed above. Following incubation, the macrophages were infectedwith C. albicans at a ratio of 1:200 with respect to the macrophages.The AmB-cochleates were then added at the concentrations of 0.1, 0.01and 0.001 μg. Twenty-four hours later, the cell cultures were lysed,samples were plated onto agar plates, and colonies were counted thefollowing day.

FIG. 12 is a graph of the number of colony forming units (CFU) for theC. albicans-infected macrophages dosed with varying concentrations ofAmB-cochleates with lipid to drug ratios of 2:1, 3:1, 4:1, and 5:1. Allcochleate formulations were efficacious at killing C. albicans;

Example 6 Amphotericin B Cochleates Prepared with DMSO and Lipid:AmBRatios of 5:1, 2:1, and 1:1 w/w

Multiple batches of Amphotericin B cochleates (CAMB) were prepared withDMSO and tocopherol (Vitamin E) by the following steps. Two methods forthe removal of solvent were employed: removal of solvent by washing thecochleates and removal of solvent by dialysis of the liposomalsuspension.

-   1. Weighing and placing 100 mg of soy PS into a 50 ml pp sterile    tube with 10 ml sterile water.-   2. Vortexing the suspension for 2 minutes.-   3. Sonicating the suspension for 3 minutes.-   4. Filtering the suspension with a 0.22 um filter and pooling the    liposomes into a 50 ml tube.-   5. Weighing and placing 10 mg (5:1), 25 mg (2:1), and 50 mg (1:1) of    AmB into 4, 15 ml pp sterile tubes with 0.5 ml DMSO (1 ml DMSO for    1:1).-   6. Adding 7.5 μl (2:1), 6.0 μl (5:1), and 10 μl (1:1) of tocopherol    at 10 mg/ml to the DMSO (the concentration of the AmB will be 20    mg/ml (5:1), 50 mg/ml (2:1), and 50 mg/ml (1:1) at this time),-   7. Vortexing the solution for a few minutes until the AmB dissolved    completely.    Remainder of Method when Removing Solvent by Washing Cochleates-   1. Mixing 5 ml of liposome with 0.507 ml of the AmB/DMSO suspension,    and vortexing the sample for a few minutes.-   2. Adding 0.5 ml of 0.1M calcium solution into the suspension with    vortexing, using an eppendorf repeater pipette with a 500 ul tip and    adding 10 μl aliquots to the tube per every 10 sec.-   3. Centrifuging the suspension for 30 minutes at 9000 rpm at 4° C.-   4. Removing the supernatant from the tube and re-suspending the    pellet with wash buffer of same volume (2 mM calcium with sterile    water).-   5. Repeating steps 3 and 4. Adjusting the final volume of the    suspension to 5 ml with 2 mM calcium wash buffer.    Remainder of Method when Removing Solvent by Dialysis of Liposomes-   1. Transferring 6 ml (1:1), and 5.5 ml (2:1 and 5:1), of    AmB/DMSO/liposomes into dialysis tubes individually.-   2. Starting the removal the DMSO using dialysis by changing the    sterile water several times and leaving overnight.-   3. On the next day, transferring the AmB/liposomes into the 50 ml    sterile tubes, and saving 0.5 ml of AmB/liposomes for the HPLC    analysis.-   4. Precipitating the liposomes by adding 0.5 ml of 0.1M calcium to    each 50 ml tube of AmB/liposomes. About 6.0 ml were precipitated for    the 5:1 and 2:1 samples, and 6.5 ml were precipitated for the 1:    sample 500 μl of liposomes were saved for the HPLC assay from each    sample. The pH was about 4.0 at this point after dialysis, and was    readjusted to a pH of 5.5 to 6.0 with 1N NaOH in final preparation.    Sterilization/Stability/Storage of Preparations-   1. Stability: The final preparations from both methods were examined    under a microscope, and the pH (about 5.5) was confirmed.-   2. Sterility: Samples of each preparation were streaked on a    chocolate plate to check the sterilization of the final preparation,    and incubated at 37° C., 4° C., and room temperature for 24 hrs, 48    hrs, and 72 hrs.-   3. Storage: The samples, treated with nitrogen and covered with    parafilm, were stored a 4° C.

The above formulations can be summarized as follows.

TABLE 4 CAMB Formulations Name of Ratio of Amount Amount of AmB 0.1MFinal Conc. Of Sample PS:AmB(w/w) of AmB soy PS Tocopherol liposomeCalcium AmB (mg/ml) AmB/DMSO 2:1 25 mg 50 mg 75 μg 5.5 ml 0.5 ml  ≈5mg/ml (washing) AmB/DMSO 5:1 10 mg 50 mg 60 μg 5.5 ml 0.5 ml 1.52 mg/ml(dialysis) AmB/DMSO 2:1 25 mg 50 mg 75 μg 5.5 ml 0.5 ml ≈3.8 mg/ml(dialysis) AmB/DMSO 1:1 50 mg 50 mg 100 μg  6.0 ml 0.5 ml ≈7.1 mg/ml(dialysis)

The recovery of AmB was determined using HPLC assay.

Results

-   1. Macroscopic observations: yellowish suspension with some settles    on the bottom of the tubes.-   2. Microscopic observations: aggregated and individual cochleates    were observed.-   3. Addition of EDTA: liposomes formed upon addition of EDTA-   4. Images of cochleate: FIG. 13 is a series of images of the 5:1 AmB    cochleates (top two panels) and the cochleates after addition of    EDTA (bottom two panels).-   6. Recovery: HPLC analysis indicated that following amounts of AmB    encochleated for each formulation indicated.    -   2:1 (Washing)=>81%    -   5:1 (Dialysis)=>91%    -   2:1 (Dialysis)=>92%    -   1:1 (Dialysis)=>92%-   7. Outcome: For the mouse study described in Example 7, the    following amounts of each formulation were set aside:    -   2:1 (Washing)=>0.2 mg/ml×2.5 ml, 14 bottles    -   5:1 (Dialysis)=>0.2 mg/ml×2.2 ml, 14 bottles (using 2^(nd)        batch)    -   2:1 (Dialysis)=>0.2 mg/ml×2.5, 14 bottles    -   1:1 (Dialysis)=>0.2 mg/ml×2.5 ml 14 bottles

Example 7 Efficacy Studies in Mice

The formulations of Example 6 were administered to mice to study theefficacy of the formulations to protect mice from a lethal dose ofCandida albicans, and to clear the organs of C. albicans in thesurviving mice.

Six groups of 10 mice were studied. The mice were administered 10⁶ cellsC. albicans intravenously through the tail vein. Starting 24 hourspost-infection, the following compositions were administered to eachmouse once daily at 2 mg/kg orally for 14 days, except for the controlgroup, which remained untreated.

-   -   a. Control Group (untreated)    -   b. AmB/deoxycholate    -   c. CAMB 2:1 (Washed)    -   d. CAMB 5:1 (Dialysis)    -   e. CAMB 2:1 (Dialysis)    -   f. CAMB 1:1 (Dialysis)        Appearance and behavior was monitored each day of the study.        Tissue burdens of C. albicans were determined in kidney, liver        and lungs for each animal at the end of the study, and colony        counts were taken. Organs were obtained and weighed,        homogenized, dilutions in buffer made, and aliquots plated onto        plates and colony counts of fungus taken several days later.        Summary of Results

FIG. 14 is a graph of the survival data for the C. albicans-infectedmice untreated or dosed daily for 14 days with AmB/deoxycholate (AmB/D),or AmB cochleates with a lipid to drug ratio of 5:1 (dialysis), 2:1(dialysis), 1:1 (dialysis), or 2:1 (wash).

FIG. 15 is a chart of the average number of C. albicans cells/gram oftissue in the liver, kidney, and lungs of C. albicans-infected miceuntreated (control), or dosed daily for 14 days with AmB/deoxycholate(AmB/D), or AmB-cochleates with lipid to drug ratios of 5:1 (dialysis),2:1 (dialysis), 1:1 (dialysis), or 2:1 (washing).

Seventy percent of control (untreated) animals died and showed hightissue burdens, while all four cochleate formulations were effective inpreventing mortality and reducing fungal cell burdens in target organs(kidneys, lungs and liver). The 5:1 (dialysis) formulation appeared moreeffective than the others in clearing the liver completely. The 2:1(washed) and 2:1 (dialysis) formulations had nearly the same efficacy.All formulations (excepted for 1:1 (dialysis) formulation) reduced thefungal cell burden as well as or better than the AmB/deoxycholateformulation. Overall, the data are consistent with effective oraldelivery of AmB from cochleates.

Example 8 Efficacy of Cochleates in Cells

The relative efficacy of the compositions of Example 6 were studied inJ774A.1 macrophages to compare the relative efficacy of the cochleatecompositions against Candida albicans.

Macrophages were seeded into a 96-well plate and incubated overnight asdescribed above. Following incubation, the macrophages were infectedwith C. Albicans at a ratio of 1:200 with respect to the macrophages.The AmB-cochleate formulations were then added at the concentrations of0.1, 0.01 and 0.001 μg/ml. Twenty-four hours later, the cell cultureswere lysed, samples plated onto agar plates, and counted the followingday.

FIG. 16 is a graph depicting the number of colony forming units (CFUs)for C. albicans-infected macrophages dosed with varying concentrations(0.1, 0.01 and 0.001 μg/ml) of AmB-cochleate formulations having lipidto drug ratios of 5:1 (dialysis), 2:1 (dialysis), 1:1 (dialysis), and2:1 (washing), or AmB/deoxycholate (AmB/D). The 2:1 (washing) and the5:1 (dialysis) formulation were the most efficacious at killing the C.albicans at 0.001 μl/ml. In contrast, the AmB/deoxycholate CFU's weretoo numerous to count at this concentration.

Example 9 Amphotericin B Cochleates Prepared with DMSO and Lipid:AmBRatio of 5:1 with and without Methylcellulose

Amphotericin B cochleates were prepared using Soy PS and DMSO withVitamin E, and a Lipid to AmB ratio of 5:1 as follows.

Preparation of Liposomes

20 ml of water was added to 200 mg of Soy-PS, vortexed for about 15minutes to form a liposomal suspension, and filtered using a 0.45 μmfilter. The suspension was sonicated for about 4 minutes and filteredagain with a 0.22 μm filter.

Addition of Cargo Moiety and Antioxidant in Solvent

2 ml of DMSO solvent was added to 40 mg of Amphotericin B. To theAmB/DMSO mixture was added 2 mg of Vitamin E and the solution wasvortexed for about 10 minutes. This solution was then added to theliposomal suspension by drop wise to addition while vortexing. The finalmixture was vortexed for about 2 minutes.

Precipitation of Cochleates

2 ml of calcium (0.1 M) was added to the liposomal suspension at a rateof 10 μl/10 s while vortexing to form cochleates.

Solvent Removal/Washing

The mixture was vortexed for about 1-2 minutes, centrifuged for about 1hour at 9000 rpm, and the supernatant was removed and replaced withfresh supernatant (water with 2 mM calcium). This washing step wasrepeated twice.

Inhibition of Aggregation

0.1%, 0.2%, 0.3%, 0.4%, or 0.5% (w/w) methylcellulose (MC) to inhibitaggregation and 0.2% (w/w) parabens to maintain sterility were added tothe suspension, and it was lyophilized to form a powder.

Images of cochleates containing 0.2%, 0.3% and 0.5% methylcellulose aregiven in FIG. 58. Particle size distributions in FIG. 59 show that theaddition of methylcellulose decreases aggregation, and that the additionof paraben slightly increases aggregation. FIG. 60 is an HPLC analysisof the cochleate showing that amphotericin B is the only compound withinthe cochleate structure.

Example 10 Efficacy Studies in Mice

AmB/deoxycholate and 5:1 AmB cochleates (CAMB) formulated as describedin Example 9 with and without 0.3% methylcellulose (MC) wereadministered to mice to study the efficacy of the formulations toprotect mice from a lethal dose of Candida albicans, and to clear theorgans of C. albicans in the surviving mice.

Six groups of 10 mice were studied. The mice were administered 5×10⁵cells C. albicans intravenously through the tail vein. Starting 24 hourspost-infection, the following compositions were administered to eachgroup once daily for 14 days in the dosage indicated, except for thecontrol group which remained untreated.

a. Control

b. AmB/deoxycholate 2 mg/kg ip

c. 5:1 CAMB (suspension) with 0.3% MC, 1 mg/kg AmB oral dosing

d. 5:1 Lyophilized CAMB with 0.3% MC, 1 mg/kg AmB oral dosing

e. 5:1 CAMB (suspension), 1 mg/kg AmB oral dosing

f. 5:1 Lyophilized CAMB, 1 mg/kg AmB oral dosing

Appearance and behavior was monitored each day of the study. On day 15,mice were sacrificed and tissue burden of C. Albicans was determined inkidney, liver and lungs for each animal. Organs were obtained andweighed, homogenized, diluted in buffer, and aliquots were plated ontoplates; colony counts of fungus were taken several days later.

FIG. 17 is a graph of the survival data for the C. albicans-infectedmice untreated or dosed daily for 14 days with AmB/deoxycholate (AmB/D),or AmB cochleates (CAMB) in suspension or lyophilized and formulatedwith or without methylcellulose (MC).

FIG. 18 is a chart of the average number of C. albicans cells/gram oftissue in the liver, kidney, and lungs of C. albicans-infected miceuntreated (control), or dosed daily for 14 days with AmB/deoxycholate,or AmB-cochleates (CAMB) in suspension or lyophilized and formulatedwith or without methylcellulose (MC).

One hundred percent of control (untreated) animals died by day 10 andshowed high tissue burdens. AmB/deoxycholate at 2 mg/kg resulted in 100%survival and completely cleared Candida from the liver and lungs anddecreased the tissue burden in the kidney by 2-3 log units. Fortypercent of the mice treated with CAMB without methylcellulose (both insuspension and lyophilized) died and both groups also showed substantialtissue burdens in target organs. However, CAMB with methylcellulose insuspension and lyophilized CAMB with methylcellulose afforded 100% and80% survival, respectively, and showed several log order reductions intissue burden relative to the other CAMB formulation. The antifungalproperties of CAMB with methylcellulose in suspension at 1 mg/kgadministered PO mimicked the behavior of AmB/deoxycholate at 2 mg/kgadministered IP. Overall, cochleates with methylcellulose showedstronger antifungal properties than cochleates without methylcellulose.

Example 11 Efficacy Studies in Cells

The relative efficacy of the compositions of Example 9 were studied inJ774A.1 macrophages to compare the relative efficacy of the cochleatecompositions (with and without methylcellulose) against Candidaalbicans.

Macrophages were seeded into a 96-well plate and incubated overnight asdescribed above. Following incubation, the macrophages were infectedwith C. albicans at a ratio of 1:200 with respect to the macrophages.The AmB-cochleates were then added at the concentrations of 5, 1, 0.1,0.01 and 0.001 μg/ml. Twenty-four hours later the cell cultures werelysed, samples were plated onto agar plates, and colonies were countedthe following day.

FIG. 61 is a graph of the number of colony forming units (CFU) for theC. albicans-infected macrophages dosed with AmB-cochleates in suspensionand lyophilized with lipid to drug ratio 5:1, with and withoutmethylcellulose. All cochleate formulations were efficacious at killingC. albicans.

Example 12 Scale Up

Amphotericin B cochleate preparation was scaled up to 5 liters using SoyPS and DMSO with Vitamin E, and a Lipid to AmB ratio of 5:1 as follows.

Preparation of Liposomes

5.4 L of water was added to 50 g of Soy-PS, vortexed for about 15minutes to form a liposomal suspension, and filtered using a 10 μmfilter.

Addition of Cargo Moiety and Antioxidant in Solvent

500 ml of DMSO solvent was added to 10 g of Amphotericin B. To theAmB/DMSO mixture was added 60 mg of Vitamin E and the solution wasvortexed for about 10 minutes. This solution was then added to theliposomal suspension by drop wise addition using a separatory funnelwhile vortexing. The final mixture was vortexed for about 2 minutes.

Precipitation of Cochleates

100 ml of calcium (0.5 M) was added to the liposomal suspension at arate of 10 μl/10 s while vortexing to form cochleates.

Solvent Removal/Washing

The mixture was vortexed for about 1-2 minutes, centrifuged for about 1hour at 9000 rpm, and the supernatant was removed and replaced withfresh supernatant (water with 2 mM calcium). This washing step wasrepeated twice.

Optional Inhibition of Aggregation

0.3% (w/w) methylcellulose (MC) to inhibit aggregation and 0.2% (w/w)parabens to maintain sterility were added to the suspension, and it waslyophilized to form a powder. Subsequently, the cochleates were treatedwith rabbit serum albumin and forced multiple times through a highpressure homogenizer such as the Avestin EmulsiFex-C5. Homogenizationpressure was maintained around 15K to 20K psi. Particle sizedistributions of cochleates before treatment with albumin, after twopasses through a homogenizer and after seven passes through ahomogenizer are shown in FIGS. 62, 63 and 64, respectively.

Example 13 Geldanamycin Cochleates

Geldanamycin (GA)-cochleates were prepared as described in Example 1.The cochleates were observed macroscopically to have successfullyencochleated GA, and also included crystals, possibly includingunencochleated GA. When cochleates were centrifuged, about one third ofthe GA was present in the supernatant. Overall, the GA was successfullyencochleated.

Example 14 Tyrphostin Cochleates

Tyrphostin AG-825 (TY)-cochleates were prepared using the solvent dripmethod described in Example 1. Good morphology of TY-cochleates wasobserved, in that it appeared that TY was successfully encochleated.

HPLC Analysis and Stability of TY-Cochleate Formulation

HPLC was used to study the stability of the TY in the cochleates, bymeasuring the concentration of TY in TY-cochleates as compared to free,i.e., unencochleated TY in solution.

FIG. 19 is a graph of the concentration of TY-cochleate preparationsversus free TY over time. As can the seen in FIG. 19, the free TYconcentration decreased to zero over time. In contrast, the TYconcentration initially dropped for the TY-cochleates (possibly due tothe degradation of free TY in the cochleate formulation), and stabilizedthereafter.

TY degrades into two products (identified as impurity 1 and impurity 2in FIG. 20). FIG. 20 is two graphs of the concentration of each impurityover time for both the free TY and TY-cochleates studied in FIG. 19.FIG. 20 confirms that the free TY degraded over time, whereas, after aninitial degradation was observed, the concentration of degradationproducts remained fairly stable for the TY-cochleates.

Biological Evaluation of Tyrphostin AG-825 Cochleates

Cytotoxicity of TY-cochleates was studied in a SKOV3 cell line (FIG.21). The TY-cochleates showed slightly higher cytotoxicity against thecancer cell line than that from free TY. The data for empty (drug free)cochleates also is shown.

Results

Tyrphostin AG-825 was successfully formulated into cochleates using themethod of the present invention. Stability tests demonstrated thatTY-cochleates have a superior stability as compared to free TY insolution, and biological analysis of TY-cochleates indicated that itdelivered similar cytotoxic effects on SKOV3 cell line to that of itsfree form in solution.

Porphyrin-cochleates also have been successfully made with ethanol, DMGand THF solvents.

Example 15 Porphyrin Cochleates

Porphyrin cochleates were prepared with Zinc Tetra-Phenyl Porphyrin(ZnTPP) and DMSO as described in Example 1, adjusted for a lipid toZnTPP ratio of 20,000:1 w/w.

The particle size and fluorescence of: plain liposomes; liposomes withZnTPP; and cochleates with ZnTPP, were evaluated and the followingresults were obtained.

TABLE 5 Particle Size and Fluorescence Particle Size Particle SizeFluorescence Intensity Mean (nm) StD (nm) Max (nm) (a.u.) Liposomes300.1 132.4 Liposomes + 280.1 122.9 595.5 45338 ZnTPP Cochleates + <10μm 596 43589 ZnTpp

FIG. 22 is an image of ZnTPP in solution (100% DMSO), and theZnTPP-cochleates. The ZnTPP in solution was dark purple, and thecochleate formulation was only slightly colored (pink), indicating thatthe ZnTPP was successfully incorporated into the cochleates, which arewhite.

FIG. 23 is a series of phase contrast images (left panels) andfluorescence images (right panels), of the ZnTPP-cochleates (top panels)and ZnTPP-liposomes (bottom panels) formed. These images indicate thatthe ZnTPP was successfully associated with the liposomes andsuccessfully encochleated.

Comparison to Cochleates Formed without Solvent

FIG. 24 is a series of phase contrast images (left panels) andfluorescence images (right panels), of ZnTPP-cochleates (top panels) andZnTPP-liposomes (bottom panels) formed without the presence of solvent.FIG. 24 indicates that the ZnTPP did not successfully associate with theliposomes or cochleates in the absence of solvent.

Interaction of ZnTPP-Cochleates with SKOV3 Cells

In order to study any interaction of the cochleates with cells,ZnTPP-cochleates and free ZnTPP (in solution with DMSO) were introducedto SKOV3 cell cultures, and imaged with fluorescence under a confocalmicroscope.

FIG. 25 is a series of images of the SKOV3 cell culture with the ZnTPPcochleates at 1 hour and 24 hours. The images demonstrate uptake of theZnTPP-cochleates into the perinuclear region, and that the ZnTPP had notsignificantly degraded at 24 hours.

FIG. 26 is a series of images of the SKOV3 cell culture with the freeZnTPP (in DMSO) at 1 hour and 24 hours. The images indicate high uptakeof the ZnTPP solution at one hour, but significant degradation at 24hours. The appearance and distribution of ZnTPP is different than thatobserved with the ZnTPP cochleates of FIG. 25.

Study of ZnTPP-Cochleates Using a Lipid Imaging Agent

Cochleates were prepared with and without ZnTPP as described above,except that prior to introduction of the ZnTPP/solvent, liposomes wereformed with 1% diolyl phosphatidylethanolamine (DOPE) liganded to pyrene(purchased Avanti). The DOPE was incorporated into the soy PS liposomesby dissolving both DOPE and lipid in solvent, drying the solvent to afilm, and using an aqueous solution to form liposomes. Confocal imageswere taken at 1 and 24 hours after introduction to SKOV3 cells to studythe uptake and any difference in the cellular distribution ofcochleate/lipid and the ZnTPP. The study of distribution was possiblebecause DOPE-pyrene fluoresces blue, and ZnTPP fluoresces red. ZnTPP inthe cochleates/liposomes appears pink.

FIG. 27 is a series of images of the SKOV3 cell culture with the emptycochleates (including DOPE-pyrene lipid) at 1 hour and 24 hours. Theseimages indicate uptake of the empty cochleates by the cells.

FIG. 28 is a series of images of the SKOV3 cell culture with theZnTPP-cochleates (including DOPE-pyrene lipid) at 1 hour and 24 hours.These images indicate uptake of the cochleates by the cells at both 1and 24 hours. The images also indicate a redistribution of lipid andZnTPP in the cells at 24 hours versus 1 hour. It appears that a portionof the ZnTPP has separated from the cochleates at 24 hours.

Together, FIGS. 27 and 28 indicate high uptake of cochleates by thecell, and subsequent release of porphyrins from the cochleates in thecell. The Figures also indicate that, once inside the cell, theporphyrin is more stable in the cochleate than free.

Example 16 Preparation of NSAID Cochleates

Acetaminophen Cochleate Preparation

Acetaminophen and DOPS were mixed in a sterile, polypropylene tube witha rubber policeman. TES buffer was added to the tube to disperse themixture in a ratio of 10 mg lipid/ml. The cochleates were formed by theslow addition (10 μL) of calcium chloride (0.1M) to the suspension ofliposomes at a molar ratio of lipid to calcium of 2:1 with an externalexcess of 6 mM calcium and then stored at 4° C. in the absence of light.

Acetaminophen cochleates were formulated with and without aggregationinhibitor, casein, which was added to the buffer solution prior to theaddition of calcium chloride in a casein to lipid ratio of 1:1 byweight.

Images were taken of the cochleates formed with (FIG. 37A, left panel)and without (FIG. 37A, right panel) the aggregation inhibitor. Asdemonstrated by the images, cochleates formed in the presence of caseindid not aggregate as did the cochleates formed without the aggregationinhibitor.

Aspirin Cochleate Preparation

Aspirin and DOPS were solublized in chloroform in a lipid/aspirin molarratio of 10:1 in a sterile glass tube. The sample was blown down withnitrogen to form a film. The sample was then resuspended in TES buffer,pH 7.4, at a ratio of 10 mg lipid/ml. The cochleates were formed by theslow addition (10 μL) of calcium chloride (0.1M) to the suspension ofliposomes at a molar ratio of lipid to calcium of 2:1 with an externalexcess of 6 mM calcium and then stored at 4° C. in the absence of light.

Aspirin cochleates were formulated with and without an aggregationinhibitor, casein, which was added to the buffer solution prior to theaddition of calcium chloride in a casein to lipid ratio of 1:1 byweight.

Images were taken of the cochleates formed with (FIG. 37B, left panel)and without (FIG. 37B, right panel) the aggregation inhibitor. Asdemonstrated by the images, cochleates formed with the aggregationinhibitor did not aggregate as did the cochleates without theaggregation inhibitor, which formed needle-like structures.

SUMMARY

The introduction of an aggregation inhibitor to the cochleates loadedwith a variety of cargo moieties inhibited cochleate aggregation. Allcochleate formulations with casein were significantly smaller thancochleates made without casein, and these cochleates were stable for atleast two months with no noticeable aggregation. Cochleates formedwithout casein aggregated over time.

Addition of Methylcellulose

Aspirin or acetaminophen cochleates with and without casein wereprepared as described above, except that liposomes were filtered througha 0.45 μm filter followed by a 0.22 μm filter, which resulted inunilamellar liposomes which contained the drug. Calcium chloride wasadded to the liposomes as also described above. Methylcellulose insuspension (0.5% of entire formulation) was added and the sample wasvortexed.

The addition of methylcellulose at this particular concentration did notreverse aggregation, but rather inhibited further aggregation ofcochleates. The size of cochleates subsequent to addition ofmethylcellulose was observed to remain stable.

Example 17 Inhibition of Edema in Rat Paw

A carrageenan model was employed to study the effect ofanti-inflammatory cochleates on edema in rat paws. Various aspirincochleates of the invention were used to treat carrageenan-induced ratpaw edema. These results were compared to free aspirin and indomethacinand empty cochleates to determine the efficacy of encochleatedanti-inflammatory drugs. Additionally, rats in all groups were examinedfor gastric irritation.

Samples Tested

-   -   1. Control (no treatment)    -   2. Indomethacin, 6 mg/kg    -   3. Aspirin control, 150 mg/kg    -   4. Large, empty cochleates    -   5. Small, casein cochleates    -   6. Large aspirin cochleates, 45 mg/kg    -   7. Large aspirin cochleates, 150 mg/kg    -   8. Small, casein aspirin cochleates, 45 mg/kg    -   9. Small, casein aspirin cochleates, 140 mg/kg    -   10. Large casein aspirin cochleates with 0.1% Vit E, 45 mg/kg    -   11. Large casein cochleates with 0.1% Vit E

Samples 6-10 were prepared in accordance with Example 16, except thatthe lipid:aspirin molar ratio was 5:1 and soy PS was used instead ofDOPS. Samples 4, 5, and 11 were prepared in a similar manner in theabsence of a cargo moiety. In all cases, water was used in lieu of TESbuffer. All samples were given by oral gavage at 1 hour prior toinjection of carrageenan on Day 0 in a volume of 3 mL of 0.5%methylcellulose. Methylcellulose at this concentration served tostabilize the cochleates but did not significantly affect the size ordistribution of the standard or casein cochleates. Volumes of rat paw(in ml) were measured prior to carrageenan injection using asemi-automated plethysmograph (Buxco). At 0 time, 0.1 ml of 1%carrageenan in 0.9% pyrogen free saline was injected into the right hindpaw of the rat. The paw volumes (in ml) were measured again 3 hours postcarrageenan injection to determine inhibition of paw edema. Four ratsfrom each of groups 2, 3, 6, 7, 8, 9 and 10 and one rat from each ofgroups 1, 4, 5 and 11 were bled (intravenously, jugular vein,approximately 1 ml blood) at 30 minutes and 4 hours post drugadministration. Blood was collected in heparinized vacutainers. 4 hourspost carrageenan injection, the stomachs of rats from all test groupswere removed after euthanasia by CO₂ asphyxiation to observe for gastricirritation (i.e. bleeding and ulcerations).

Inhibition of rat paw edema for all-samples is presented in FIG. 38. Ingeneral, the control group and the groups given cochleates notcontaining aspirin show no decrease in the level of edema in the ratpaw. Large aspirin cochleates (cochleates not made with an aggregationinhibitor) show a decrease in edema only slightly larger than that offree aspirin or indomethacin. Small cochleates (with an aggregationinhibitor), however, show a significant decrease in edema in comparisonto both free aspirin and indomethacin and large aspirin cochleates.Additionally, large aspirin cochleates with vitamin E show more of adecrease in edema when compared to plain large aspirin cochleates.

FIG. 39 shows the incidence and level of severity of gastric irritationproduced by samples 2, 3, 7 and 9. In general, indomethacin produced thegreatest gastric irritation, followed by unencochleated aspirin. Aspirincochleate formulations produced less incidence of irritation whencompared to both aspirin and indomethacin.

Example 18 Activation of Macrophages by Anti-Inflammatory Cochleates

To examine the effects of cochleates on lipopolysaccharide (LPS) plusIFN-γ induced NO production, J774A.1 macrophages were treated with LPSplus IFN-γ in the presence and absence of empty cochleates. Macrophageswere also treated with and without empty cochleates in the absence ofLPS plus IFN-γ.

Since NO production requires the enzymatic activity of NOS, its activitywas measured by NO secretion using the method of Griess (nitrite).Briefly, 100 μl of sample was reacted with the Griess reagent at roomtemperature for 10 minutes. Amount of NO₂ was then determined bymeasuring the absorbance at 540 nm in a microplate reader. The standardcurve was obtained using the known concentration of sodium nitrite. Inall experiments, NO₂ concentration in wells containing medium only wasalso measured as a blank control.

FIG. 40 indicates that empty cochleates (EC) are immunologically inert.That is, they neither enhance nor inhibit NO production induced by LPSplus IFN-γ at all concentrations assayed. In contrast, the addition ofLPS plus IFN-γ to the macrophages with and without empty cochleatesresulted in a dramatic increase in iNOS production. In addition, allconcentrations of the empty cochleates showed no sign of cellulartoxicity as was observed under phase contrast microscopy.

In order to determine the in vitro efficacy of anti-inflammatorycochleates of the invention, J774A.1 mouse macrophages were incubatedwith LPS (1 μg/ml) plus IFN-γ (10 μg/ml) in the presence or absence ofstandard aspirin cochleates and acetaminophen cochleates prepared asdescribed in Example 16, free aspirin, free acetaminophen and emptycochleates (control) for 15 hrs.

As shown in FIG. 41, standard cochleates containing aspirin andacetaminophen exhibited greater in vitro efficacy than free aspirin andacetaminophen at inhibiting NO production.

Example 19 Particle Size Analysis of Cochleates of the Invention

Cochleates stabilized with 1% casein were evaluated with the N4 plusfrom Coulter. Briefly, 20 μl of the suspension of empty cochleatesprepared in accordance with Example 17 (without NSAIDs) were added to2.5 ml of D.D. H₂O. The samples were equilibrated over 20 minutes. Thesamples were then analyzed for 2 minutes at a 90° angle. Two differentpopulations of cochleates were observed, one centered at 25 nm, and theother one at 350 nm (FIG. 34A). The population centered at about 25 nmlikely consists of casein micelles and not cochleates. Any such micellescan be removed, e.g., by centrifugation.

The particle size of aggregated, standard cochleates was also evaluatedusing the LS230 from Coulter. 100 μl to 200 μl of the sample was addedto 250 ml of washing buffer in the vessel until the PIDS (PolarizationIntensity Differential Scattering) reached 45%. The duration time for arun was 120 s and the number of cycles was 3. Four differentpopulations, one centered at approximately 1 μm, one at approximately 10μm, one at approximately 30 μm and the last one at approximately 50 μmwere observed. (FIG. 34B)

Example 20 Preparation of Cochleates with Various Aggregation Inhibitors

Rhodamine labeled phosphatidyl ethanolamine (Rho-PE) liposomes wereprepared by adding di-oleoyl-PS (DOPS) and Rho-PS to chloroform at aratio of 10 mg lipid/ml solvent. The DOPS was present at 0.1% or 0.01%of the total lipid. The sample was blown down under nitrogen to form afilm. Once dry, the sample was resuspended in a TES buffer at a ratio of10 mg lipid/ml buffer. The liposomes were then passed through a 0.22 μLfilter. The homogeneous population of Rhodamine labeled liposomes werestored at 4° C. in the absence of light under nitrogen.

Sterile glass tubes, each containing 100 μl fluorescent Rhodaminecochleates in TES buffer were prepared Cochleates were formed by theaddition of 10 μl aliquots of 0.1M calcium chloride until a molar ratioof lipid to calcium of 2:1 and an external excess of 6 mM calcium wasreached. 10 μl Half and Half was added to one tube and vortexed for 4minutes. Whole milk, at a 1:1 ratio of whole milk to lipid, was added toa second tube and vortexed for one minute. Evaporated fat free milk, ata 1:2 weight ratio of evaporated milk to lipid was added to a third tubeand vortexed for 4 minutes. A fourth tube was used as the control, andas such, no aggregation inhibitor was added.

FIGS. 35A-D are four fluorescent images of the Rhodamine-labeledcochleates obtained. The Figures demonstrate the effect of formulatingcochleates, in the presence of various aggregation inhibitors: half andhalf (FIG. 35A), whole milk (FIG. 35B), and fat-free milk (FIG. 35C).FIG. 35D is an image of the control composition of cochleates that donot include an aggregation inhibitor.

FIG. 36 depicts the aggregated cochleates prior to the addition of milk(left image) and after the addition of milk (right image). These imagesindicate that milk caused aggregation to reverse.

Example 21 Uptake of Cochleates by Macrophages

Rhodamine labeled phosphatidyl ethanolamine (Rho-PE) cochleates wereprepared as described in Example 20, except that casein was added to theformulation prior to the addition of calcium in a casein to lipid ratioof 1:1. Additionally, no milk products were added to the casein-coatedRho-PE cochleates.

Sterile cover slips were placed in the wells of 24-well plates. J774.1macrophages were harvested as described above, counted using ahemacytometer and seeded at a concentration of 1×10⁵, and allowed toincubate overnight to ensure adherence. Rhodamine-PE cochleates werethen added at a final concentration of 50 μg lipid/ml and 5 μg lipid/ml.

The cover slips were removed at the desired time point, generally aboutone hour, rinsed in DMEM to remove any free cochleates, placed invertedon microscope slides and observed for uptake using phase contrast andfluorescence microscopy. Standard cochleate formulations were observedto remain within the macrophage for several days and slowly transfer thefluorescent lipid from endocytic vessels throughout the rest of themacrophage. In contrast, the nanocochleates were only observed up to36-hours after administration. Due to their size and/or altered surfacecharacteristics, the cochleates with casein were taken up moreaggressively than standard cochleates (without casein) by culturedcells. Nearly every cell incubated with the cochleate compositionprepared with casein showed intracellular fluorescence, indicating thatthe cochleates were rapidly taken up by the macrophages (FIG. 33B). Incontrast, standard cochleates were not taken up as aggressively by themacrophages as is shown by intracellular fluorescence (FIG. 33A).

Example 22 Cochleates Formed with Protonized Vancomycin

Cochleates Formed with and without Calcium

Vancomycin cochleates are expected to increase the oral bioavailabilityof vancomycin while limiting its side effects. Vancomycin cochleateswere formed with and without calcium.

14.6 mg of Dioleoyl Phosphatidylserine (DOPS, Avanti, Ala.) was used asthe starting lipid material for each cochleate formulation. Thephospholipid powder and 7.6 mg protonized Vancomycin (Vanco) powder weremixed in a molar ratio of 4.3:1.1 mL of modified TES buffer (2 mM TES,150 mM NaCl, 2 mM L-Histidine), adjusted at pH 3 was added to eachmixture. To one formulation, calcium also was added. The mixture wasvortexed for 2 minutes.

Optical microscopy, using phase contrast technique, revealed thepresence of cochleates in both the formulation without calcium (FIG.42), and the formulation with calcium (FIG. 43). The cochleates werecentrifuged at 3000 rpm at 4° C. for 20 min. The content of Vanco in theaggregates was assessed by OD absorption at 282 nm with aspectrophotometer. Results showed that the lipid associated with theVanco such that the vancomycin comprised about 40% of the precipitate byweight for the formulation without calcium and about 70% of theprecipitate for the formulation with calcium.

Addition of EDTA chelating agent to the formulation with calciumresulted in a rapid transformation of the cochleate into openedstructure (FIG. 44), suggesting that the cochleates included stackedsheets of lipid bilayer and cationic drug.

Cochleates Formed with Alternative Acidification Step

Vanco crystals were added to preformed DOPS liposomes (FIG. 45A). Thevanco was solubilized as TES buffer (pH 7.4) was added to disperse themixture in a ratio of 10 mg lipid/ml. HCl (0.1N) was used to bring thepH to 5.0 or 6.5, at which point an association of the Vanco with thelipid were visible under the microscope. The protonized Vanco wasobserved to associate with the negatively charged bilayer surface. 10 μLof calcium chloride (0.1M) then was slowly added to the suspension ofliposomes at a molar ratio of lipid to calcium of 2:1 with an externalexcess of 6 mM calcium and then stored at 4° C. in the absence of light.

Cochleates Formed with and without an Aggregation Inhibitor

Vancomycin cochleates were formulated with an acidification step asdescribed above with and without an aggregation inhibitor (casein),which was added to the buffer solution prior to the addition of calciumchloride in a casein to lipid ratio of 1:1 by weight.

Images were taken of the cochleates formed with (FIG. 45B) and without(FIG. 45C) casein. When EDTA was added to the cochleates, they wereopened to form liposomes as shown in FIG. 45D. The efficacy of thecochleates against Staph. aureus was studied in vitro as described inExample 24, below.

Example 23 Cochleates Formed with Tobramycin

Cochleates Formed with Acidification Step

Tobramycin crystals were added to pre-formed liposomes (FIG. 48A).Tobramycin was solubilized as TES buffer (pH 7.4) was added to dispersethe mixture in a ratio of 10 mg lipid/ml. HCl (0.1N) was used to bringthe pH to 5.5, at which point an association of the tobramycin with thelipid were visible under the microscope. The protonized tobramycin wasobserved to associate with the negatively charged bilayer surface. 10 μLof calcium chloride (0.1M) was slowly added to the suspension at a molarratio of lipid to calcium of 2:1 with an external excess of 6 mM calciumand then stored at 4° C. in the absence of light.

Cochleates Formed with and without an Aggregation Inhibitor

Tobramycin cochleates were formulated with and without an aggregationinhibitor (casein), which was added to the buffer solution prior to theaddition of calcium chloride in a casein to lipid ratio of 1:1 byweight.

Images were taken of the cochleates formed with (FIG. 48B) and without(FIG. 48C) casein. EDTA was added to the cochleates of FIG. 48C and thecochleates were observed to open as shown in FIG. 48D. The efficacy ofthe cochleates against Staph. aureus was studied in vitro as describedin Example 24, below.

Example 24 Bactericidal Activity of Cochleates

J774A.1 is a well characterized murine macrophage-like cell line thathas been extensively used to study Staphylococcal aureus-macrophageinteractions. The J774A.1 cells were maintained at −80° C. prior to useand were prepared for the phagocytosis assays as described above.

J774A.1 macrophages were counted using a hemacytometer, seeded into96-well plates and incubated overnight. Following incubation, themacrophages were infected with Staphylococcal aureus or Pseudomonasaeruginosa at a ratio of 1:200 with respect to the macrophages.

Free Vanco and Vanco cochleates prepared with and without casein asdescribed in Example 22, were added to the macrophages infected withStaph. A. at concentrations of 1, 5, 10, and 25 μg/ml.

Free tobramycin and tobramycin cochleates prepared as described inExample 23 with and without casein were added to the macrophagesinfected with P. aeruginosa and P. aeruginosa alone at concentrations of1, 5, 10, and 25 μg/ml.

Following incubation for 3 and 6 hours, the plates were removed andobserved. Medium was removed and replaced with 100 μl cold sterilewater. The plates were incubated 10 minutes, at which point the 100 μlcold sterile water was pipetted vigorously to disrupt the cellularmembrane. 25 μl of this suspension was placed onto Sabouraud DextroseAgar plates, and placed in a dry incubator overnight at 37° C.Staphylococcal aureus or Pseudomonas aeruginosa colony forming units(CFU's) were counted the to following day.

FIGS. 46 and 47 are graphs demonstrating the efficacy data for the Vancocochleates (with and without casein) against Staphylococcal aureusversus free vancomycin at 3 and 6 hours after administration,respectively.

FIGS. 49 and 50 are graphs demonstrating the efficacy of the tobramycincochleates of the invention (with and without casein) againstPseudomonas aeruginosa, versus free tobramycin at 3 and 6 hours afteradministration, respectively.

As FIGS. 46, 47, 49 and 50 indicate, the cochleates of the inventionincrease the effectiveness of the cargo molecule against bacteria incells. Additionally, vancomycin and tobramycin cochleates including anaggregation inhibitor show a significant increase in efficacy inrelation to both free drug and cochleates formed without aggregationinhibitor.

Example 25 Caspofungin Cochleates

Cochleates Formed with Calcium—Solvent Drip Method

Soy phosphatidylserine (Soy PS, Degussa) was used as the starting lipidmaterial for each cochleate formulation. 100 mg soy PS was mixed with 10mL water or saline, and the mixture was vortexed, forming liposomes. 10mg of protonized caspofungin (5 mg for 20:1 cochleates or 20 mg for 5:1cochleates) was then dissolved in 1 mL DMSO. The DMSO solution wasslowly added to the liposomal solution. After the caspofungin/soy PSliposomal solution was mixed, 1.5 mL of 0.1M calcium chloride solutionwas added at a rate of 10 μl/10 s in order to precipitate a solid.Resulting formulations, along with observations about cochleatemorphology are presented in Table 1, below.

Cochleates Formed with Calcium—Aqueous Method

Numerous formulations using the aqueous drip method were prepared usingvarying combinations of starting materials. Soy phosphatidylserine (SoyPS, Degussa) and DOPS were both used as the starting lipid material forcochleate formulations using the aqueous drip method. Soy PS or DOPS(100 mg) was mixed with 5 mL water, saline, or buffer and the mixturewas vortexed until liposomes formed. Protonized caspofungin (10 mg for10:1 cochleates, 20 mg for 5:1 cochleates) was then dissolved in 5 mLwater, saline, or buffer. The caspofungin solution was added slowly tothe liposomal solution. After mixing, 1.5 mL of a 0.1 M calcium chloridesolution was added to the caspofungin/soy PS liposomal solution at arate of 10 μl/10 s in order to precipitate a solid caspofungincochleate. Resultant formulations, along with observations aboutcochleate morphology and measurements of “free” caspofungin arepresented in Table 1, below.

TABLE 1 Caspofungin cochleate formulations “free” PS:caspo PSBuffer/saline/ Morphology caspo- Method (w/w) source water (+/−EDTA)fungin DMSO 20:1 soy PS water or saline OK ND drip 10:1 soy PS water orsaline OK ND  5:1 soy PS water or saline OK ND aqueous 10:1 DOPS waterOK 15%  drip 10:1 DOPS saline OK 4% 10:1 soy PS water OK 2% 10:1 soy PSTES buffer OK 2% 10:1 soy PS saline OK 0.1%   5:1 soy PS saline OK 1%*“free” caspo indicates caspofungin which did not precipitate with thesoy PS.Briefly, the morphology of formulations made with both the solvent dripmethod and the aqueous drip method were indicative of cochleatestructures. That is, they both demonstrated an opening to liposomes uponaddition of EDTA. Additionally, it appears that the use of salinediminished the amount of free caspofungin in comparison to the use ofwater when cochleates were formulated using the aqueous drip method.Cochleates Formed with Additional Acidification Step

100 mg soy PS was combined with 5 mL saline, and the mixture wasvortexed until liposomes formed. 50 mg protonized caspofungin was thencombined with 5 mL saline buffer at pH 5.5. This pH ensured that thecaspofungin remained protonized and multivalent. The caspofunginsolution was slowly added to the soy PS liposomes. Cochleates began toform immediately upon addition of caspofungin because of the highvalency of the protonized moiety.

These cochleates were also formed with additional calcium, and withsterile water instead of saline. These three formulations were thenmaintained at 4° C. for five days in order to test the stability of theresultant cochleates. HPLC analysis of the cochleates and thesupernatant was completed to measure concentration of caspofungin inboth, and is summarized in FIG. 56. It is shown that in sterile water,the concentration of caspofungin in the cochleate gradually decreases,while the concentration of caspofungin not associated with a cochleate(“free caspofungin”) increases. This decrease of caspofungin in thecochleates and increase in free caspofungin can also be observed for thesaline formulation with excess calcium. The formulation with salineonly, however, remained stable over the five day period.

Acidification of Solution Subsequent to Cochleate Formation

Caspofungin cochleates formed with an alternative acidification step (pH5.5) as described above were subsequently treated with varying amountsof sodium hydroxide and hydrochloric acid in order to vary the pH from 1to 9 (pH tested with litmus paper). Phase contrast micrographs at pH 1,4, 6, 7, 8, and 9 are depicted in FIG. 57. It appears that theformulations at pH 4 and pH 6 are cochleates, but open to form liposomeswhen the pH is raised to 9.

Addition of Bovine Serum Albumen to Caspofungin Cochleates

The particle size distribution of 10:1 and 5:1 PS:caspofumgin cochleateswas measured using diffraction-based light-scattering from 0.5 to 500microns with Beckman-Coulter LS230. FIGS. 53A and 53B (Vanco) are twographs depicting the particle size distributions of 10:1 PS:caspofumgincochleates and 5:1 PS:caspofungin cochleates, respectively. The 10:1PS:caspofungin formulation was then treated with bovine serum albumin(BSA), followed by C-5 homogenization in order to reduce the meanparticle size of the cochleates. Once again, the particle sizedistribution of the caspofungin cochleates was measured. FIG. 54 depictsthe particle size distribution of the caspofungin cochleates, anddemonstrates the decrease in particle size upon homogenization andaddition of the BSA.

Example 26 Characterization of Caspofungin Cochleates

Caspofungin cochleates with a lipid:caspo ratio of 10:1 and 5:1formulated as described in Example 25, using saline (0.9% NaCl), withadditional Vitamin E (1% w/w, Roche) added at the liposomal stage, werecharacterized physically and chemically.

Morphology

The morphology of 10:1 and 5:1 caspofungin cochleates was observed usingphase contrast microscopy and are shown in FIGS. 51A and 52A,respectively. EDTA was also added to the caspofungin cochleates of thesame formulations in order to observe the opening of the cochleatestructures. Phase contrast micrographs are given in FIGS. 51B and 52B,and show the cochleates opening to form liposomes.

Chemical Characterization

Concentrations of caspofungin in both the supernatant (to determine“free” caspofungin) and in the pellet (encochleated caspofungin) weremeasured using HPLC (FIG. 55), and the concentration of soy PS wasdetermined using a modified Bartlett P_(i) assay. The PS:caspofunginratio in the cochleates was determined using these values. Theconcentration of free caspofungin in both the 10:1 formulations and the5:1 formulations ranged from approximately 0.7% to approximately 8.2% oftotal caspofungin. A sterility test for bacterial growth on agar plateswas also investigated, and it was determined that all formulationspassed.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A cochleate composition comprising: Amphotericin B; aplurality of cochleates comprising soy phosphatidyl serine; and0.2%-0.4% (w/w) methylcellulose; and wherein the weight ratio of soyphosphatidyl serine to Amphotericin B is about 5:1.
 2. The compositionof claim 1, wherein the plurality of cochleates has a mean diameter ofless than about 600 nm.
 3. The composition of claim 1, wherein theplurality of cochleates has a mean diameter of less than about 500 nm.4. The composition of claim 1, wherein the size distribution of theplurality of cochleates is less than about 700 nm.
 5. The composition ofclaim 1, wherein the size distribution of the plurality of cochleates isless than about 550 nm.
 6. The composition of claim 1, wherein thecomposition is in the form of a nasal spray.
 7. A pharmaceuticalcomposition comprising a therapeutically effective amount of thecochleate composition of claim 1 and a pharmaceutically acceptablecarrier.
 8. A method of treating a subject that can benefit from theadministration of Amphotericin B, comprising the step of: administeringthe pharmaceutical composition of claim 7 to said subject, such that thesubject is benefited.
 9. The method of treatment according to claim 8,wherein the administration is by a mucosal or a systemic route.
 10. Themethod of treatment according to claim 8, wherein the administration isa mucosal route selected from the group consisting of oral, intranasal,intraocular, intrarectal, intravaginal, topical, buccal andintrapulmonary.
 11. The method of treatment according to claim 8,wherein the administration is intranasal.
 12. The method of treatmentaccording to claim 11, wherein the pharmaceutical composition isdelivered in a form selected from the group consisting of a spray, anebulae, a mist, an atomized vapor, an irrigant, an aerosol, a wash, andan inhalant.
 13. The method of claim 8, wherein the pharmaceuticalcomposition is used to treat rhinosinusitis.
 14. The method of treatmentaccording to claim 8, wherein the administration is by a systemic routeselected from the group consisting of intravenous, intramuscular,intrathecal, subcutaneous, transdermal and intradermal.
 15. The methodof claim 8, Amphotericin B is administered to treat-parasitic disorders.16. The method of claim 8, wherein the pharmaceutical composition isadministered to treat asthma.
 17. The method of claim 8, wherein thepharmaceutical composition is administered to treat at least onedisorder selected from the group of consisting of asthma, chronicrhinosinusitis, allergic fungal sinusitis, sinus mycetoma, non-invasivefungus induced mucositis, and non-invasive fungus induced intestinalmucositis.